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Mice carrying a null mutation of the Lrfn4 gene (Lrfn4–/– mice) have been used to investigate in vivo functions of SALM3 (Li et al., 2015). These mice show reduced excitatory synapse number, as supported by spontaneous excitatory synaptic transmission and electron microscopic data, but their inhibitory synapses are minimally affected. However, NMDAR-mediated synaptic transmission and NMDAR-dependent synaptic plasticity—both LTP and LTD (long-term depression)—were unaffected in Lrfn4–/– mice.

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SALM3 has recently been implicated in the regulation of epilepsy (Li et al., 2017). This study showed that SALM3/Lrfn4 expression is significantly increased in two distinct animal models of epilepsy, and further found that suppression of SALM3 expression by virus-mediated SALM3 knockdown ameliorates seizure activity as well as neuronal hyperexcitability. These results suggest that SALM3 promotes epileptogenesis and that its suppression has therapeutic potential.

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Whether SALM4 regulates synapse development or function has remained unclear, partly because SALM4 does not have synaptogenic activity (unlike SALM3 and SALM5) or a PSD-95-binding C-terminal tail (unlike SALMs1–3). However, it should be noted that SALM4 is immunodetected in neuronal synapses in addition to dendrites and axons (Seabold et al., 2008). A recent study using mice lacking SALM4 (Lrfn3–/– mice) demonstrated that SALM4 has unexpected negative effects on the density of excitatory and inhibitory synapses (Lie et al., 2016). Specifically, Lrfn3–/– mice display increases in the number of excitatory and inhibitory synapses in the hippocampus, as supported by the density of PSDs and frequency of mE/IPSCs.

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The strong influence of Lrfn4 KO on excitatory synapse development relative to synaptic function or plasticity is in line with the role of SALM3 as a synapse organizer that regulates presynaptic differentiation by interacting with LAR-RPTPs. SALM3/Lrfn4 was found to associate with 14-3-3 and NCK signaling adaptors to regulate actin-rich lamellipodial structures in monocytes through mechanisms involving the Rac1 small GTPase (Konakahara et al., 2011, 2012). Given that dendritic spines are actin-rich structures (Cingolani and Goda, 2008), LAR-RPTP–induced SALM3 clustering at postsynaptic sites might promote 14-3-3/NCK- and Rac1-dependent actin polymerization to promote synapse development.

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Behaviorally, Lrfn4–/– mice show reduced locomotor activity in both novel and familiar environments, but exhibit normal anxiety-like behaviors. These mice also perform normally in learning and memory tests, including Morris water maze, novel object recognition, contextual fear conditioning, and T-maze spontaneous/reward alternations. The minimal effect of Lrfn4 KO on learning and memory behaviors is in keeping with the largely normal LTP in these mice. However, it remains unclear how Lrfn4 KO leads to behavioral hypoactivity.

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This study further addressed the mechanisms underlying the SALM4-dependent negative regulation of synapse density, reporting that postsynaptic SALM4 interacts in cis with SALM3, which possesses synaptogenic activity and exhibits a highly overlapping distribution pattern in the brain (Mah et al., 2010; Lie et al., 2016). This cis-interaction, in turn, inhibits the trans-synaptic interaction of SALM3 with presynaptic LAR-RPTPs and suppresses SALM3-dependent presynaptic differentiation (Lie et al., 2016). In support of these conclusions, coexpression of SALM4 with SALM3 in heterologous cells blocks binding of purified soluble LAR to SALM3 and inhibits SALM3-dependent presynaptic differentiation in contacting axons of cocultured neurons. Given that the transmembrane domain of SALM4 is required for cis-interactions with and inhibition of SALM3, it is unlikely that LRR-mediated SALM4–SALM3 heterodimerization, if it occurs, underlies the cis-inhibition.

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Importantly, genetic support for these conclusions is provided by SALM3/SALM4 double-KO mice, in which the increased excitatory synapse density observed in SALM4 single-KO mice is normalized, as supported by both electron microscopy and mEPSC recordings (Lie et al., 2016). In contrast, double KO does not normalize the increased density of inhibitory synapses, suggesting that SALM4 negatively regulates inhibitory synapses through mechanisms independent of SALM3. Because SALM4 can also interact in cis with SALM5, which possesses synaptogenic activity (Mah et al., 2010), SALM5 might also play a role in SALM4-dependent regulation of inhibitory synapses. In support of this possibility, expression of SALM3 and SALM5 in heterologous cells and cultured neurons induces both excitatory and inhibitory presynaptic contacts, and SALM5 knockdown in cultured neurons suppresses both excitatory and inhibitory synapses (Mah et al., 2010). However, whereas SALM3, artificially aggregated on dendritic surfaces of cultured neurons, induces secondary clustering of PSD-95, aggregated SALM5 does not induce gephyrin clustering (Mah et al., 2010).

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SALMs have been implicated in diverse neurodevelopmental and psychiatric disorders (Table 3). LRFN2, encoding SALM1, has recently been implicated in learning disabilities, as supported by impaired working memory and executive function in three individuals in a family with a 6p21 autosomal dominant microdeletion (~870 kb) encompassing three genes, including LRFN2 (Thevenon et al., 2016). Similarly, levels of SALM1/LRFN2 proteins were found to be substantially decreased in postmortem brains of patients with neurodegenerative disorders associated with cognitive declines such as Alzheimer’s disease and Parkinson’s disease with dementia (Bereczki et al., 2018). In addition, single nucleotide polymorphism (SNP) analyses have linked LRFN2 with the risk of antisocial personality disorder (Rautiainen et al., 2016). More recently, a targeted gene sequencing strategy identified missense mutations of LRFN2 in individuals with ASD (Morimura et al., 2017), as noted above.

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LRFN5, encoding SALM5, has been frequently associated with ASDs. SNP analyses have linked a chromosomal locus on 14q21.1 between FBXO33 and LRFN5 to a risk for ASD (Wang et al., 2009). A balanced de novo t(14;21)(q21.1;p11.2) translocation that leads to a ~10-fold reduction in the expression of LRFN5, located ~2 Mb from the translocation breakpoint, was identified in a 19-year-old girl with autism and intellectual disability (de Bruijn et al., 2010). In addition, a genome-wide association study showed that LRFN5 is associated with a risk for ASDs (Connolly et al., 2013). Similar results were reported in a whole-exome sequencing study, although the association score was not high (De Rubeis et al., 2014). More recently, family-based diagnostic exome sequencing identified a point mutation (p.V572X) in LRFN5 in an individual with ASD (Farwell Hagman et al., 2017).

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LRFN5 has also been implicated in other neurodevelopmental and psychiatric disorders. For example, an ~890-kb deletion encompassing LRFN5 exons was identified in a girl with developmental delay, learning disability, seizures, microcephaly and receding forehead by high-resolution array comparative genomic hybridization (Mikhail et al., 2011). In addition, a high-resolution linkage analysis identified LRFN5 among schizophrenia-related copy number variations (Xu et al., 2009). Lastly, a recent genome-wide gene- and pathway-based analysis identified LRFN5 as one of four depression-associated genes (Nho et al., 2015). It is unclear why LRFN5 is frequently associated with brain disorders. Although its synaptogenic activity might be a contributor, the fact that SALM3, another synaptogenic SALM, is not closely associated with brain disorders suggests against this possibility. Studies using transgenic mice lacking Lrfn5 may provide insight into this question.

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Since the discovery of the SALM/Lrfn family about a decade ago, a large number of studies have elucidated basic characteristics and functional features of SALMs. Recent reports have shed additional light on the properties and functions of SALMs, identifying novel presynaptic ligands (LAR-RPTPs) of SALM3/5, resolving the crystal structures of SALMs in complex with LAR-RPTPs, elucidating the in vivo functions of SALMs, and revealing clinical implications of SALMs, collectively helping to better understand the functions of this protein family. However, our understanding of SALMs remains at a relatively early stage, leaving a number of questions to be explored.

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For example, although SALMs can form heterodimers, and SALM dimers can explain the reported heteromeric cis-interactions between SALMs in the brain, it remains unclear whether SALM family members other than SALM5 and SALM2 form dimeric structures. It is also unclear whether SALMs directly interact with NMDA/AMPARs and, if so, whether these interactions regulate receptor functions or synaptic adhesions in a reciprocal manner. Because SALM3 and -5 are part of many LAR-RPTP–interacting postsynaptic adhesion molecules, whether SALM3/5 has its own unique roles, or redundant functions, remains to be determined.

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In vivo functions of SALMs also require further exploration. It will be interesting to determine whether SALMs have distinct functions in different brain regions and cell types. Because individual SALMs have largely unique cytoplasmic regions, SALM-associated synaptic signaling pathways are likely to be quite diverse. Circuit mechanisms underlying various behavioral phenotypes of Salm-KO mice, in particular those associated with SALM-related developmental and psychiatric disorders, also need to be investigated. Given the rapid increase in information on the biology and pathophysiology of SALMs, the next 10 years are likely to witness a dramatic increase in our understanding of this interesting family of synaptic adhesion molecules.

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The development of a cure for HIV infection is a major public health objective . Despite the ability of antiretroviral therapy (ART) to significantly reduce disease-related morbidity and mortality in HIV-1 infection, viral reservoirs persist indefinitely in latently infected cells . HIV persists during ART primarily within circulating and tissue-resident, long-lived memory CD4+ T cells that harbor integrated HIV DNA; these cells are not cleared with ART and are a source of viral rebound when treatment is discontinued . A major HIV eradication strategy involves aborting the initial seeding of these long-lived reservoirs by the very early initiation of ART . For example, initiation of ART in a perinatally infected infant at 31 hours of life led to significant reductions in the viral reservoir and a significant time off ART (>2 years) before eventual viral recrudescence . However, the impact of extremely early ART on HIV persistence and seeding the viral reservoir with the potential to prevent establishment of lifelong infection in adults is unknown.

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Antiretroviral drugs initiated before HIV exposure (pre-exposure prophylaxis [PrEP]) can be an effective method of preventing HIV acquisition [8–11]. PrEP programs involve HIV antibody testing before the initiation of prophylactic ART in individuals at high risk for acquiring HIV. PrEP is typically started following negative HIV antibody or combined antibody/antigen screening in the absence of clinical symptoms . Because there is a delay between HIV infection and when an HIV antibody or combined antibody/antigen test is reactive, PrEP may be unknowingly started in an individual who has very recently been infected with HIV. As a result, a small number of individuals may begin 2-drug ART just prior to or after the development of detectable plasma HIV-1 RNA (the transition from the "eclipse phase" to Fiebig stage I of infection) and prior to the detection of HIV antigen or antibody . PrEP programs are therefore ideal settings in which to identify individuals treated extremely early during infection for the longitudinal study of HIV-1 reservoir persistence in blood and various tissues .

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The aims of this study were to determine the impact of extremely early initiation of ART on the size of the HIV reservoir in blood and various tissues and the potential for long-term ART-free remission. As a result, we studied 2 PrEP study participants who initiated ART during emergent, unrecognized HIV infection in Fiebig stage I, with 1 individual treated just as he was transitioning out of the “eclipse phase” of HIV infection . We describe the results of extensive tissue and blood sampling in these individuals and the result of a highly monitored treatment interruption. Using this case and our previously described recipient of an allogeneic bone marrow transplant (hematopoietic stem cell transplantation [HSCT] Participant B) , we also describe potential biomarkers for HIV reactivation during prolonged states of viremia post-ART interruption.

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The PrEP Demo Project was a prospective study of PrEP for men who have sex with men (MSM) in which participants were tested for HIV both by HIV antibody/antigen combination assay and by HIV RNA on the day of PrEP initiation . There was no prespecified plan for the present analysis at that time. Two participants were identified in the study who had positive viral load tests performed on the day of initiation of PrEP (truvada/emtricitabine). PrEP was converted to standard ART once HIV infection was confirmed. The participants were enrolled in the longitudinal University of California San Francisco (UCSF) SCOPE study after providing written informed consent. The study was approved by the UCSF Committee on Human Research. A protocol and an analysis plan were in place prior to analytical treatment interruption (ATI). The protocol and STROBE checklist are included in the supporting information (S1 Protocol and S1 STROBE Checklist).

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To more fully explore biomarkers associated with HIV reactivation after the interruption of ART, we also accessed peripheral blood mononuclear cells (PBMCs) from a previously described case of an HIV-infected adult who underwent an allogenic hematopoietic stem-cell transplantation (HSCT Participant B) . All HIV reservoir studies for HSCT Participant B during ATI were conducted at commercial laboratories and cryopreserved PBMCs were not available.

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PBMCs and plasma were collected longitudinally either by large-volume peripheral blood draws or by leukapheresis and purified by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation. Colorectal and ileal tissue were collected and processed as previously described . A whole, inguinal lymph node was obtained from each participant by surgical excision for mononuclear cell isolation and downstream HIV reservoir characterization months following combination ART initiation. Total PBMCs, purified CD4+ T cells, or CD4+ T cell subsets [naive (TN), central memory (TCM), transitional memory (TTM), and effector memory (TEM)] from blood and tissues were collected via bead-based separation (Stem Cell Technologies) or by fluorescence-activated cell sorting using previously described methods . Bone marrow biopsies were performed, followed by sorting and collection of 4 cell populations: myeloid cells (CD33+Lin+/−), CD3+CD4+ T cells (CD33−Lin+CD4+), multilineage CD4+ cells (CD33−Lin−CD4dim), and Lin− cells that are not CD4+ or myeloid cells (CD33−Lin−CD4−CD34+/−). Cerebrospinal fluid (CSF) was collected by lumbar puncture and centrifuged to separate the liquid and cellular fraction for downstream HIV-1 detection, and quantification was carried out as previously described .

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HIV-1 RNA was isolated from baseline (pre-PrEP) and subsequent plasma samples followed by single-genome sequencing (SGS) of HIV-1 protease-pol (pro-pol) or a 1.5 kb portion of the envelope gene as previously described . Population sequencing on subsequent timepoints was performed using TRUGENE (Siemens, Tarrytown NY). PBMCs and tissue-derived cells were analyzed for HIV-1 persistence using a variety of highly sensitive assays in up to 10 independent laboratories located at the UCSF, University of Montreal, University of Pittsburgh, University of California San Diego (UCSD), and Johns Hopkins University. Assays utilized were previously described and included highly sensitive quantitative PCR for total and integrated HIV-1 DNA (unspliced and total RNA) , droplet digital PCR (ddPCR) (HIV pol DNA and 2-LTR circles) , total virus recovery assay, quantitative viral outgrowth assay (qVOA) , Tat/Rev Induced Limiting Dilution Assay (TILDA) , and whole HIV genome sequencing . In addition, large volume plasma was tested using the MEGA iSCA .

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CD4+ T cells obtained by leukapheresis from both participants approximately 18 months following the initiation of ART were tested using the murine (humanized mouse) viral outgrowth assay (mVOA) as previously described . Briefly, 530 million and 379 million CD4+ T cells were divided and injected intraperitoneally into 8–10 humanized female young adult NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice from Jackson Labs (53 million and 50 million cells per mouse from the 2 participants). Plasma HIV-1 RNA testing was then performed following up to 5.5 weeks after engraftment on blood obtained by weekly mandibular sinus bleed not exceeding 0.5% of body weight. Mice were euthanized by CO2 inhalation and HIV-1 sequencing was attempted from plasma and spleen cells using cDNA and methods optimized for low HIV-1 RNA copy numbers . The Johns Hopkins University Institutional Animal Care and Use Committee approved this research and it was conducted in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals within fully AAALACi accredited animal facilities. Mice were group-housed with other mice xenografted from the same donor in microisolator caging (Allentown) and fed a commercial rodent chow (Harlan) and hyperchlorinated water ad libitum.

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A carefully monitored ATI was performed on 1 participant (PrEP Participant A) who initiated PrEP an estimated 10 days following HIV infection and had no definitive HIV detected in blood or tissues for 32 months on ART. After ATI, viral load testing was performed twice weekly using the COBAS AmpliPrep/COBAS TaqMan V.2 assay (Roche) for the first 3 months, followed by weekly testing thereafter with close clinical observation. Large-volume blood draws were also obtained monthly for the isolation of PBMCs, and plasma was obtained for further reservoir and flow cytometric testing. ART was initiated immediately after confirmation of detectable plasma HIV-1 RNA.

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Flow cytometric testing was performed on cryopreserved PBMCs isolated longitudinally just before, during, and after ATI for PrEP Participant A and HSCT Participant B. Thawed PBMCs were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific), Brilliant Violet 711-conjugated anti-CD4 (SK3) (BD Biosciences), Brilliant Violet 650-conjugated anti-CD3 (SP34-2) (BD Biosciences), allophycocyanin (APC)-conjugated anti-CD38 (HB7) (BD Bioscience), PE-conjugated anti-CD30 (BERH8) (BD Biosciences), APC-H7-conjugated anti-HLA-DR (L243) (BD Bioscience), CD8 Alexa Fluor 700-conjugated anti-CD8 (RPA-T8) (BD Bioscience), BV786-conjugated anti-CD16 (3G8) (BD Bioscience), PE-Cy7-conjugated anti-CD107a (H4A3) (BD Bioscience), FITC-conjugated anti-CD56 (NCAM 16.2) (BD Bioscience), and PerCP-conjugated anti-CD69 (L78) (BD Bioscience). Cells were then analyzed on a BD LSRII flow cytometer (BD Biosciences). Single stained beads (Life Technologies) were used for compensation, and fluorescence minus one (FMO) controls were used to set gates. Data for phenotyping were acquired on all events and analyzed in FlowJo V10 (TreeStar). Examples of gating strategies are shown in S1 Fig.

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CD4+ and CD8+ T cell phenotypes were further characterized on live cells for PrEP Participant B by flow cytometry after exclusion of dead cells (Fixable Aqua dye, Molecular Probes) using the following fluorescently labeled antibodies: CD3 BV650 clone SK7, CD4 BV711 clone OKT4, CCR7 BV785 clone G043H7, CD45RA APC-Cy7 clone H100, Tbet PE clone 4B10, Eomesodermin PE-e610, PD-1 BV421 or BV605 clone EH12.2H7, CD160 PE-Cy7 clone BY55, Ki-67 FITC clone Ki-67, Granzyme B FITC clone GB11, Perforin PE-Cy7 clone B-D48 (all Biolegend), and CD8α APC-R700 clone RPA-T8 (BD Biosciences). Positive gates for activation markers and effector cell transcription factors were drawn based on expression of these markers in peripheral blood naive CD8+ T cells isolated from an HIV-uninfected donor. To limit experimental variability, flow cytometry was performed on the same day.

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After thawing, PBMCs were incubated at 37°C overnight at a concentration of 2 × 106 cells/mL in RPMI medium containing 10% FBS and penicillin/streptomycin. The next day, 1 × 106 cells were stimulated with Gag pooled peptides (final concentration 1 μg/mL in DMSO provided by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 Con B Gag Peptide Pool cat #12425), for 6 hours in the presence of brefeldin A and monensin (BD). The percentage of CD8+ or CD4+ T cells producing interferon gamma (PE-dazzle, clone 4S.B3, Biolegend), tumor necrosis factor alpha (Alexa fluor 700, clone mAb11, eBioscience) or interleukin-2 (BV785, clone MQ1-17H12, Biolegend), or degranulation as detected by CD107a expression (APC, clone H4A3, Biolegend), was measured by flow cytometry, and the frequency of positive cells was determined after subtraction of the frequency measured in wells incubated with DMSO alone. Examples of gating strategies are shown in S2 Fig.

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Graphical analyses were performed using Prism v.6 (GraphPad software). Mathematical modeling of reservoir size, rebound rate, and probability of cure were performed using our previously described methods . A summary of these methods and the input variables used is provided in S1 Text.

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Participant A, a 54-year-old male, was HIV-uninfected at 2 PrEP pre-enrollment visits but continued to have ongoing sexual risk for HIV infection. He then initiated tenofovir/emtricitabine PrEP and usage was confirmed by testing drug levels. Seven days after PrEP initiation, results from the baseline (day 0 of PrEP) visit revealed a plasma HIV RNA level of 220 copies/mL (Abbott RealTime HIV-1, lower limit of detection [LLD] <40 copies/mL), and 69 copies/mL by the Single-Copy Assay (LLD 1 copy/mL); 4th generation EIA (Abbott) and rapid HIV-antibody (Clearview HIV 1/2 Stat-Pak) tests were negative. Based upon these results (and a negative pooled HIV RNA, 4th generation EIA, and rapid HIV-antibody tests at 2 pre-enrollment visits 21 and 13 days prior), it was determined that he had been in the transition from the eclipse phase to Fiebig stage I HIV infection (estimated 10 days after HIV infection ) at the time of initiating PrEP. PrEP was substituted with a 4-drug ART regimen (darunavir/ritonavir/raltegravir/tenofovir/emtricitabine) 7 days after the initiation of PrEP. This ART regimen was chosen due to concern about the potential for the development of drug resistance to tenofovir/emtricitabine. The participant was asymptomatic at the time. HIV western blot assays (BioRad) were repeatedly indeterminate (p55 only) and became nonreactive at an estimated 130 days after time of infection. SGS from the plasma sample from the date when PrEP was initiated confirmed that the individual was infected with subtype B virus without known drug resistance mutations.

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Plasma HIV RNA levels subsequently declined following PrEP and combination ART initiation: 220 copies/mL (PrEP baseline), 120 copies/mL (7 days after initiation of PrEP), and <40 copies/mL but detectable (estimated 22 days after starting PrEP). All subsequent plasma HIV RNA levels were undetectable. Low-level cell-associated HIV RNA (3.2 copies/million CD4+ T cells) was detected 32 days after infection. However, sorted rectal CD4+ T cells were negative for HIV RNA and DNA (collected 1.9 months after infection), and leukapheresis-collected PBMCs enriched for total CD4+ T cells and sorted CD4+ T cell subsets (TN, TCM, TTM, and TEM) were negative for cellular HIV RNA, total HIV DNA (confirmed in 2 independent laboratories), integrated HIV DNA, and 2-LTR circles (collected 2.1 months after infection). Over the following 2 years, no further HIV (nucleic acid, viral outgrowth, total virus recovery, or whole genome sequences) could be detected, despite sampling from ileum, rectum, lymph nodes, bone marrow, CSF, and circulating CD4+ T cell subsets. A detailed timeline of clinical viral load measurements and results of longitudinal HIV-1 reservoir assays are shown in Fig 1 and Table 1. Chemokine receptor 5 (CCR5) genotyping revealed that the individual did not carry any CCR5 Δ32 mutations, and HLA typing revealed that he was HLAB5701 negative. We estimated that greater than 1 billion CD4+ T cells were eventually examined without any evidence of HIV infection.

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No HIV was detected in colorectal and lymph node tissues, bone marrow, CSF, plasma, and very large numbers of PBMCs obtained longitudinally from Participant A (top panel) who started PrEP 10 days following HIV infection. In contrast, low-level HIV RNA and DNA were intermittently detected in PBMCs from Participant B (bottom panel) who initiated PrEP 12 days following infection prior to starting a 4-drug ART regimen. One of 10 humanized mice given 53 million input cells from Participant A developed low-level HIV RNA in plasma (201 copies/mL), but sequencing of HIV from plasma was unsuccessful. Abbreviations: ABC, abacavir; CSF, cerebral spinal fluid; ddPCR, droplet digital PCR; DTG, dolutegravir; FTC, emtricitabine; GALT, gut-associated lymphoid tissue; iSCA, HIV integrase single copy plasma RNA assay; PBMC, peripheral blood mononuclear cell; PrEP, pre-exposure prophylaxis; qVOA, quantitative viral outgrowth assay; RAL, raltegravir; r/DRV, ritonavir-boosted darunavir; TDF, tenofovir; TILDA, Tat/Rev Induced Limiting Dilution Assay; VOA, viral outgrowth assay.

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Three months following infection, Participant B had very low level detectable HIV-1 RNA (2.9 copies/106 cell) and DNA (14.3 copies/106 cells) in TTM and TEM CD4+ T cell subsets, respectively, and again 2.4 and 5.5 HIV RNA copies/106 total CD4+ T cells approximately 9 months after infection. No HIV could be detected from CSF, bone marrow cells or CD4+ T cell subsets from a whole excised inguinal lymph node 8–9 months following infection. No definitive HIV outgrowth could be detected with a qVOA performed 32 months after infection utilizing 20 million input total CD4+ T cells. Very low levels of HIV-1 RNA (<50 copies/mL) were detected from several of the viral outgrowth assay (VOA) wells, but no increases were observed in RNA over time, and no bulk or single genome HIV-1 sequences could be obtained (see Fig 1 and Table 2).

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Abbreviations: ART, antiretroviral therapy; c/mL, copies/mL; GALT, gut-associated lymphoid tissue; LOD, limit of detection; Neg, negative; PBMC, peripheral blood mononuclear cells; PrEP, pre-exposure prophylaxis; qVOA, qVOA, quantitative viral outgrowth assay; UCSF, University of California San Francisco.

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The percentage of CD8+ and CD4+ T cells expressing CD30 and CD69 are shown in (A) and (B). The percentage of CD16+ and CD56+ NK cells expressing CD30 and CD69 are shown in (C) and (D). T cell expression of CD30 from the allogeneic HSCT recipient who also underwent ATI is shown in (E). Abbreviations: ATI, analytical treatment interruption; c/mL, copies/mL; HSCT, hematopoietic stem cell transplantation; NK, natural killer; PrEP, pre-exposure prophylaxis.

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Sufficient cells were available for further immunological phenotyping from PrEP Participant A, and we identified an increase in the frequency of eomesodermin expressing CD8+ T cells prior to viral rebound. However, no patterns emerged during ATI for expression of PD1, TIGIT, or Ki67. There were no changes in CD8 responses in CD107a expression, and no significant intracellular INF-gamma, TNF-alpha, or IL1 production was detected in response to overlapping HIV-1 gag peptide pools prior to, during, or following ATI.

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Abbreviations: ART, antiretroviral therapy; ATI, analytical treatment interruption; c/mL, copies/mL; CSF, cerebrospinal fluid; ddPCR, droplet digital PCR; GALT, gut-associated lymphoid tissue; HSPC, hematopoietic stem and progenitor cells; iSCA, integrase single copy assay; LOD, limit of detection; LTR, long-terminal repeat; Neg, negative; PBMC, peripheral blood mononuclear cell; qVOA, quantitative viral outgrowth assay; TILDA, Tat/Rev Induced Limiting Dilution Assay; UCSD, University of California San Diego; UCSF, University of California San Francisco; VOA, viral outgrowth assay.

clinical case

PrEP Participant B, a 31-year-old male, was HIV-uninfected at a pre-enrollment visit 6 weeks before initiating tenofovir/emtricitabine PrEP but had ongoing sexual risk for HIV infection. On the date of initiation of PrEP his Clearview HIV 1/2 Stat-Pak, HIV-1 CMIA (Abbott Ag/Ab combo assay), HIV-1 IFA, western blot, and HIV 1/2 MultiSpot Rapid Test were all non-reactive. Six days after initiation of PrEP, results from the baseline (day 0 of PrEP) visit revealed a plasma HIV of 359 copies/mL and PrEP was discontinued. Based on these results and clinical information, it was determined that he was infected approximately 12 days prior to starting PrEP (Fiebig stage I). Eight days after starting PrEP, the plasma HIV-1 RNA level increased to 668 copies/mL. HIV genotyping was positive for the M184M/I resistance mutation (no mutation was detected by genotyping on PrEP day 0), and 3,343 copies/mL were measured 9 days after the initiation of PrEP. He started combination ART (darunavir/ritonavir/raltegravir/tenofovir/emtricitabine) 12 days after starting PrEP, with subsequent decline of HIV plasma RNA to 351 copies/mL and 39 copies/mL on post-PrEP days 16 and 44, respectively. All subsequent plasma HIV RNA levels were undetectable starting at post-infection day 91. He changed ART to tenofovir/emtricitabine/dolutegravir/rilpiverine 56 days after starting PrEP and then to abacavir/lamivudine/dolutegravir 29 months following initial PrEP (Fig 1). HIV antibody testing remained negative through day 219 after the initiation of PrEP, but a western blot was indeterminate (p24 weekly positive, all other bands negative) on day 258.

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Given the very low level or lack of detectable HIV-1 from large numbers of cells from various tissues, large quantity plasma, or CSF, we performed a leukapheresis on both participants and used large numbers of CD4+ T cells for input into a previously reported mVOA . We obtained 530 million total CD4+ T cells from Participant A approximately 18 months following infection on ART. One of 10 mice given 53 million input cells developed low-level HIV RNA in plasma (201 copies/mL) following in vivo activation with anti-CD3 antibody approximately 5.5 weeks after engraftment, but HIV was not detected in spleen tissue (<1 million spleen cells present). However, HIV-1 RNA and cDNA sequencing at an independent laboratory was unsuccessful from plasma and spleen cells, and true infection in cells from Participant A could not be verified. We obtained 500 million total CD4+ T cells from Participant B. Three of 8 mice developed detectable HIV RNA starting approximately 4.5 weeks following cell transfer (1,000; 5,000; and 11,000 copies/mL, respectively, from a total of 50 million CD4+ T cells per mouse).

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Following 34 months of continuous ART, Participant A provided informed consent to undergo a carefully monitored ATI (Fig 2). The patient remained clinically well with undetectable plasma viral load measurements through 225 days of observation post-interruption. On day 225, he had a detectable, low-level plasma HIV-1 RNA of 36 copies/mL. Repeat testing 6 days later (day 231) confirmed rebound with virus increasing to 77,397 copies/mL. He initiated therapy (tenofovir/emtricitabine/ritonavir/darunavir/dolutegravir) on post-ATI day 231 prior to receiving the confirmatory test. He then had viral loads of 158 and 19 copies RNA/mL on days 245 and 252, respectively. Subsequent plasma viral load testing revealed no detectable HIV RNA, and he has remained clinically well. Single genome sequences of a 1,190 base-pair region of HIV-1 Pol were monoclonal and identical to the PrEP baseline sequences. The level of intra-sequence diversity was extremely low at both time points (<0.02%) but distinct from the consensus ancestral subtype B sequence. HIV-1 envelope single genome (1,557 base-pair region) analysis at the time of recrudescence revealed 2 unique but highly related sequences; HIV envelope sequences were not obtained at PrEP baseline (S3 Fig).

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We used a collection of mathematical modeling approaches (see S1 Text) to better understand the dynamics of reservoir seeding and rebound in these participants. For PrEP Participant A, the estimated latent reservoir size immediately before treatment interruption, based only on the observed time of rebound (226 days) was 0.0020 [0.00045, 0.0063] infectious units per million cells (IUPM) (brackets give 95% credible intervals). With this estimated reservoir size (including uncertainty), about 1% of identical individuals (i.e., having similar restrictions in reservoir size) would be expected to achieve lifelong (>70 year) ART-free HIV remission. These results are consistent with estimates of reservoir size from various assays described in Fig 1 and Tables 1 and 2. Based on the viral load level at initial diagnosis and a calibrated model of reservoir seeding during acute infection , we estimate that the reservoir size prior to the initiation of ART was 0.02 IUPM, with about 1 log uncertainty in either direction. qVOA results on day 166 suggest that there is a 95% probability that the reservoir size is below 0.075 IUPM . If we assume the positive outgrowth in the mVOA is a true positive signal, then there is a 95% probability that the reservoir size is below 0.0057 IUPM. If we assume the outgrowth was not real, then the central estimate for the reservoir size is 0.0020 IUPM (95% credible interval 0.00028–0.014 IUPM). Assuming there are around 1011 total body CD4+ T cells , these estimates collectively suggest there were only approximately a few hundred cells infected with replication-competent HIV provirus prior to treatment interruption. Separately, we estimated the exponential growth rate of viral load during rebound to be 1.3/day, very similar to that seen in the HSCT Boston participants and near the central value seen in acute infection [41–43], but much higher than that seen in rebound following chronic infection (excluding the prolonged time off ART prior to first detection of HIV-1) [44–46].

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Flow cytometric characterization of surface markers of T and natural killer (NK) cell activation was performed on samples from PrEP Participant A before and during ATI and following HIV rebound in order to identify potential predictors of viral recrudescence. In addition, pre- and post-ATI samples were investigated from HSCT Participant B, an individual who lost detectable HIV-1 in blood and gut tissue following allogeneic HSCT for malignancy. Similar to PrEP Participant A, the HSCT participant experienced confirmed HIV rebound 225 days after interrupting ART and had similar exponential growth rate during recrudescence . Interestingly, surface expression of CD30, a member of the tumor necrosis factor (TNF) super-receptor family and lymphoma tumor marker, increased on CD4+ and CD8+ T cells months prior to detectable plasma HIV in both of these participants (Fig 3). The frequency of CD30+ and CD69-expressing cells also increased on CD4+ and CD8+ T cells and CD56+ and CD16+ NK cells prior to viral recrudescence in PrEP Participant A. Overall, increases in the frequency of CD30-expressing cells appeared to be larger than those expressing CD69. However, no distinct patterns in lymphocyte HLA-DR/CD38 were observed in either participant, and only CD30 expression increased prior to rebound in the HSCT recipient.

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It is likely that there are rare individuals who start PrEP during the true eclipse phase, when HIV has the potential to establish a long-lived reservoir but before the development of detectable HIV in blood or tissues. Detecting a potential “cure” in such a setting will be challenging but such efforts are ongoing. Of note, non-human primate studies have demonstrated that, while ART initiated within 48 hours of SIV challenge is able to prevent infection [50–52], ART started 3 days following SIV infection leads to viral rebound following cessation of therapy . As a result, the "curative window" between infection and the potential to abort infection after exposure is likely very small. It is also possible that there may be individuals infected just before the start of PrEP, but in whom infection would not be known for some time until they stop ART or until a 2-drug regimen is unable to suppress the virus.

study

Our study is one of the first to incorporate an mVOA to measure potential HIV persistence following an intervention that leads to loss of detectable HIV in blood and various tissues. One mouse became viremic (at low levels) and 3 mice became viremic following receipt of cells from PrEP Participants A and B, respectively. This positive finding with PrEP Participant A's PBMCs may be the only evidence that this individual had persistent HIV prior to ART interruption. Unfortunately, sequence verification of plasma and splenic HIV in mice from both PrEP participants could not confirm definitive viral outgrowth from participant cells. Sample volumes are limited in murine models of HIV infection and were exhausted during testing. Nonetheless, 2 published studies suggest that mVOAs may be more sensitive than traditional outgrowth assays to detect replication-competent HIV in individuals with undetectable HIV-1 DNA or RNA by traditional means . If this is the case, our study suggests that sampling of hundreds of millions of PBMCs may, at times, be more sensitive than tissue-based studies for the detection of residual HIV infection since a much larger number of cells can be interrogated. Further studies comparing mVOAs with traditional ex vivo co-culture assays utilizing rigorous positive and negative controls are certainly warranted.

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After this delay, the rapid HIV rebound dynamics in PrEP Participant A were again similar to those observed in HSCT Participant B , consistent with rapid exponential growth seen with primary infection. Unlike the HSCT participant, however, the PrEP recipient was asymptomatic and the peak viral load may have been mitigated by the earlier reinitiation of ART. The observed rapid rebound kinetics also differ from those in individuals who achieved post-treatment HIV-1 control following early ART initiation .The rapid rebound kinetics and lack of post-treatment control observed in this study are likely secondary to the lack of HIV-specific immunity since exponential growth is lower in the setting of withdrawal of ART initiated during chronic infection when HIV-specific immunity is present. Concomitant immune-modifying therapies may be necessary in order to achieve ART-free HIV control in very-early treated individuals.

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PrEP Participant B initiated ART later than PrEP Participant A and had higher levels of viremia at his baseline visit. In many ways, he is similar to the “Mississippi baby” in that he started ART with moderate levels of viremia and subsequently had a difficult-to-detect reservoir. Individuals who initiate therapy during the earliest stages of infection will almost certainly need other “curative” interventions before ART might be interrupted with the expectation that a viral relapse will be avoided. It is also possible that higher peak plasma HIV RNA levels and subsequent low-level viral reservoir detection in samples from PrEP Participant B were a result of the appearance of an emtricitabine-resistant associated mutation and exposure to a single active antiretroviral drug during initial PrEP.

other

We report 2 cases of extremely early HIV diagnosis and initiation of ART at the threshold when plasma viremia begins to expand exponentially (the end of the so-called “eclipse phase” and beginning of Fiebig stage I). These stages of HIV infection precede the time when acute HIV infection becomes clinically apparent and are theoretically the earliest time when ART can be initiated in an adult . To the best of our knowledge, PrEP Participant A is the earliest documented case of adult HIV infection followed by immediate initiation of ART with the exception of successful post-exposure ART prophylaxis, and it would be very challenging to initiate therapy any earlier. Despite the complete or near-complete loss of detectable HIV in blood and a variety of tissues, HIV rebounded in this person 225 days following cessation of ART. Although identifying hyperacute infection is rare, our group has previously reported that 15.6% of patients referred to our HIV clinic for newly diagnosed infection had detectable plasma HIV-1 RNA but negative HIV-specific antibody test results . These data include individuals taking part in a rapid treatment initiation study and may overestimate the incidence of acute infection identified during Fiebig stage I. Excluding participants in this study, 6.3% of individuals presented with positive plasma HIV-1 RNA and no detectable HIV-specific antibodies at the time of diagnosis .

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The delayed timing of viral rebound in PrEP Participant A was similar to that observed in an HIV-infected individual who lost detectable HIV in blood and tissue following allogeneic HSCT (Boston Participant B). In each case, modeling predicted a residual HIV replication-competent reservoir of perhaps hundreds of infected CD4+ T cells throughout the body, which explains the lack of detectable HIV from blood or tissues despite massive sampling. Overall, allogeneic HSCT and extremely early ART initiation appear to have similar long-term effects on reducing both the residual HIV reservoir and immune responses. Nonetheless, small numbers of latently infected cells likely persisted in these individuals and became activated, leading to HIV rebound in the absence of ART. Modeling of these types of participants leads to wide confidence intervals, which are likely due to the varying size of the reservoir in different patients, uncertainty around model parameters, and the stochastic nature of reactivation of latently infected cells in vivo. It is also possible that a non-CD4+ T cell source of HIV persistence contributed the viral rebound; a majority of cells tested for persistence in blood and tissue were CD4+ lymphocytes. Of note, the median time to viral rebound in 8 Thai individuals treated with ART during later phases of Fiebig stage I was recently reported to be 26 days . Although sample size is limited, these and our data suggest that differences in initiation of ART time of just a few days during Fiebig stage I infection may have a noticeable impact on the duration of ART-free remission.

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A major emphasis of HIV curative science has been to identify potential markers or correlates of HIV rebound before or after treatment cessation. CD30, a member of the TNF receptor superfamily, is expressed on very small percentages of lymphocytes and myeloid cells but is dramatically upregulated on Hodgkin and other lymphoma cells [55–59]. CD30 has been implicated in the activation, proliferation, and cell death of selected cell populations ; and infections with human T-cell lymphotropic virus (HTLV), Epstein-Barr virus (EBV), and poxviruses can lead to increases in CD30 expression . In addition, increases in the plasma concentration of the soluble form of CD30 have been associated with HIV disease progression prior to ART initiation [57,58,60,64–71]. Although anecdotal, our data in PrEP Participant A and the HSCT participant suggest that upregulation of CD30 may occur prior to detectable plasma HIV-1 RNA. Furthermore, given a similar but somewhat less pronounced pre-rebound expression pattern of CD69 in the PrEP Participant A, it is possible that these markers represent more global lymphocyte activation, proliferation, or stress responses that are detectable prior to viral recrudescence. Further investigation of CD30 and related cell-surface markers as potential predictors of HIV rebound is urgently needed.

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These cases also suggest that PrEP programs should test individuals for HIV using an HIV test with the narrowest window period possible, just before initiating PrEP. If cost permits and in settings with a high incidence of acute HIV (e.g., STI clinics), it is reasonable to test with a plasma HIV RNA before PrEP initiation and when reinitiating PrEP after an interruption. Fourth generation combination p24 antigen/antibody tests will identify most patients with acute HIV but miss those in the earliest Fiebig stages, such as those presented in this analysis. Plasma HIV RNA testing should also be considered when reinitiating PrEP after interruption . PrEP programs should recommend an immediate switch to conventional ART if an individual is found to be newly HIV-positive. Potential benefits of such programmatic changes include (1) lower rates of missed acute HIV diagnoses, (2) decreased acquired drug resistance to tenofovir/emtricitabine, (3) individual clinical benefit , and (4) fewer subsequent transmission events .

study

This study was limited by its small sample size. The identification of individuals treated during hyperacute infection is rare given the rapid increase in plasma HIV-1 RNA during the early phase of Fiebig stage I infection and the fact that hyperacute infection may be asymptomatic. The limited number of individuals included in the analysis make it difficult to draw definitive conclusions about the impact of very early ART on restricting HIV reservoir size or prolonging ART-free remission. In addition, a larger number of individuals are required to validate potential cell-surface markers of HIV persistence or predictors of HIV rebound. Despite the challenges with including very early treated individuals in prospective studies, these cases provide valuable information as to the rapidity of seeding of the HIV reservoir and the impact of extremely early ART on HIV persistence.

other

In summary, we report 2 cases of extremely early initiation of prophylactic ART immediately following the “eclipse phase” in Fiebig stage I (at approximately 10 days of HIV infection). A very small residual HIV reservoir size was observed in these participants who started very early PrEP and subsequently converted to full ART. In 1 individual, we observed a prolonged period of ART-free remission similar in duration to the allogeneic HSCT Boston Participant B. However, although HIV persisted indefinitely in both of these PrEP cases, a continuum may exist across PrEP, post-exposure prophylaxis and curative early ART strategies. Further investigation in larger cohorts of individuals treated extremely early following HIV infection is warranted.

other

Other viruses may be relevant in ocular diseases, such as gammaherpesvirus and pestivirus. Antibodies against a virus from the subfamily Gammaherpesvirinae were detected in semi-domesticated reindeer in Finnmark County, Norway, with a prevalence of 3.5% in 2013 , and the presence of a novel gammaherpesvirus was also found to be circulating among semi-domesticated reindeer in Norway . Other viruses in this subfamily are associated with malignant catarrhal fever (MCF) in different wild and domestic ruminant species, which may cause keratoconjunctivitis, with ocular discharge and panophthalmitis, as seen in cattle [24, 25], or ocular discharge, keratitis and conjunctival hyphema, as observed in American Bison (Bison bison) . Serological screenings have demonstrated that pestivirus infections are enzootic in semi-domesticated reindeer in Norway [27, 28] and Sweden and the susceptibility of these animals to bovine viral diarrhea virus infection (BVDV; family Flaviviridae, genus Pestivirus) has been experimentally demonstrated . A pestivirus (V60-Krefeld; Reindeer-1), genetically related to border disease virus (BDV), was isolated from a captive reindeer in Duisburg Zoo (Germany) . However, pestivirus has not yet been isolated from wild or semi-domesticated reindeer . Some ocular signs, including ocular discharge, conjunctivitis and hemorrhages in the sclera and palpebral conjunctiva, have been reported in cattle infected with BVDV , but the possible association between pestivirus infection and IKC in reindeer is not known.

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Environmental factors, such as stress, dust, or UV light have also been proposed to contribute to the development of the disease [15, 34–36]. According to the questionnaire survey, IKC in reindeer is most often seen during September to November in connection with the collection, transport and handling of reindeer, which also coincides with the period when animals are observed more carefully and closely, and also when animals are more stressed .

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Infectious keratoconjunctivitis (IKC) is a severe transmissible ocular disease, which affects many ruminant species worldwide, including reindeer (Rangifer tarandus). In cattle (Bos taurus), infectious bovine keratoconjunctivitis (IBK) is considered the most important eye disease worldwide , and the bacterium Moraxella bovis is considered as the main causative agent . However, IBK is regarded as a multifactorial disease, to which also other bacteria, viruses and environmental factors can play a role [3, 4]. In sheep (Ovis aries) and goats (Capra aegagrus hircus), bacteria from the family Chlamydiaceae can be involved in the development of IKC . In sheep in Norway, Mycoplasma conjunctivae and possibly Moraxella (Branhamella) ovis are considered primary causative agents . IKC has also been described in several wildlife species [7, 8], e.g. chamois (Rupicapra rupicapra) [9, 10], alpine ibex (Capra ibex) and moose (Alces alces) . Many attempts have been made to isolate bacteria from ruminants during outbreaks of IKC, although it is not always obvious if the bacteria isolated have been the responsible pathogen, or if they rather represented opportunistic or environmental bacteria. It has been shown that many different species of bacteria can be cultured from the eyes of apparently healthy ruminants, with no associated clinical signs. Rehbinder and Glatthard indicated that several bacterial species could be cultured from the eyes of 86% of clinically healthy and 90% of diseased reindeer. Barber et al. and Egwu et al. also found this to be true in most clinically unaffected cows (n = 261; Scotland, UK) and 97.5% of sheep (n = 480; England, UK), respectively.

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IKC is a rather common disease in reindeer which usually affects individual animals or small groups, particularly calves and young animals, but the disease may also appear as regular outbreaks, affecting tens or hundreds of animals in a herd, and having major impact on animal welfare and reindeer herding economy . An illustrated questionnaire distributed to reindeer herders in Norway and Sweden revealed that 55.0% of the responding herders (35/63) had observed clinical signs similar to IKC the previous year (2010) . This disease has been described in reindeer for more than 100 years . IKC in reindeer is considered a multi-factorial disease. Many types of bacteria have been isolated from reindeer with IKC, such as Moraxella bovoculi, Moraxella ovis, Escherichia coli, Listeria monocytogenes or Staphylococcus sp. [18–20], which all may play a role in the development of the disease. However, from an outbreak of IKC in reindeer it was concluded that Cervid herpesvirus 2 (CvHV2) was the causative and transmissible agent, accompanied by secondary opportunistic bacterial infections and it has recently been shown experimentally that CvHV2, alone and in combination with Moraxella bovoculi, was able to cause clinical sympotms characteristic for IKC in reindeer .

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Semi-domesticated reindeer (n = 341) were sampled in Norway (n = 171), Sweden (n = 139) and Finland (n = 31) (Fig. 1) in the period 2010–2014, either during herd gatherings (live animals, n = 143) or from slaughterhouses (dead animals, n = 198).Fig. 1Distribution of sampling sites of semi-domesticated reindeer (n = 332) with names of the administrative units (siida, sameby and cooperatives, in Norway, Sweden and Finland, respectively), indicating sampling of live animals (blue squares), slaughtered animals (red circles) or both live and slaughtered animals (yellow diamond)

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Distribution of sampling sites of semi-domesticated reindeer (n = 332) with names of the administrative units (siida, sameby and cooperatives, in Norway, Sweden and Finland, respectively), indicating sampling of live animals (blue squares), slaughtered animals (red circles) or both live and slaughtered animals (yellow diamond)

review

In both cases, reindeer with clinical signs of IKC were prioritized for sampling when observed. In total, 108 animals had clinical signs related to IKC, 113 did not show such signs, and for 120 animals, information on clinical signs was not available (Table 1).Table 1Semi-domesticated reindeer sampled in 2010–2014 in Norway, Sweden and FinlandReindeer (all ages) sampled1Reindeer calves (≤1 year old) sampledReindeer adults (>1 year old) sampledOcular clinical signs bOcular clinical signs bOcular clinical signs bCountryLocationTotal012NRTotal012NRTotal012NRNorwayIfjordfjellet28-2-269-1-812-1-11Lødingen4920--29-----2020---Sennalandet23-2-216---612---12Sørreisa3591016-265813-9423-Tromsdalen36306--2727---936--SwedenKaresuando3416711-3416711------Kiruna20812--20812-------Kikkejaure3221524----------Lainiouoma Sameby33-2112-27-207-6-15-Malå Sameby5---52---23---3Rans Sameby15---1510---104---4FinlandKallioluoma cooperation109-1-109-1------Käsivarsi cooperation11911-11911------Muotkatunturi cooperation1010---1010--------341113624612019284493326752710830aThe number of reindeer (all ages) sampled includes animals from which the age information was unavailable, together with the number of calves and adults.bThe severity of the ocular disease was scored with 0 for asymptomatic animals, 1 for animals with increased lacrimation and/or mild conjunctivitis or 2 for animals with moderate to severe clinical signs of IKC. Animals from which information was not registered were placed in column NR

clinical case

bThe severity of the ocular disease was scored with 0 for asymptomatic animals, 1 for animals with increased lacrimation and/or mild conjunctivitis or 2 for animals with moderate to severe clinical signs of IKC. Animals from which information was not registered were placed in column NR

clinical case

The age of the animals was determined by reindeer owners. Animals born in the previous calving season and therefore under 1 year of age were registered as calves (n = 192). Animals born in previous calving seasons were registered as adults (n = 75), and for 74 animals, age-class information was not available.

other

For the animals with clinical information available, severity of the ocular disease was scored from 0 to 2, with 0 for asymptomatic animals, 1 for animals with increased lacrimation and/or mild conjunctivitis or 2 for animals with moderate to severe clinical signs of IKC, including corneal and periorbital oedema, conjunctivitis, keratitis, pus with or without blood in the eye or its surrounding area, corneal ulcus or collapse and fibrosis of the eye.

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Blood samples were collected from the jugular vein in blood tubes with and without K2EDTA (BD Vacutainer®; BD, Plymouth, UK), using a venoject needle (Terumo, Leuven, Belgium) for live animals, or by collecting blood directly into open tubes during bleeding of slaughtered animals. Tubes were centrifuged for 10 min at 3.000 g to prepare serum or plasma and were then stored at −20 °C until analysis.

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Swab samples for virology (Applimed SA, Châtel-St-Denis, Switzerland) were inserted into the conjunctival fornix, rubbed gently against the conjunctival mucosa and placed in sterile cryotubes with 800 μl of Eagle’s Minimum Essential Medium (EMEM) with antibiotics (final concentrations of 100 IU/ml of penicillin, 100 μg/ml of streptomycin, 50 μg/ml of gentamicin and 2.5 μg/ml amphotericin B) and stored at −80 °C until analysis.

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Swab samples for bacteriology were obtained from the conjunctival fornix as described for the virology swabs and placed in Amies transport medium with charcoal (Transwab® Amies Charcoal Transport; MWE, Wiltshire, England), transported unfrozen to the laboratory and cultured within 2–9 days after sampling, depending on transport time to the laboratory.

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Serum samples were tested for the presence of antibodies against alphaherpesvirus with a commercial BoHV1 blocking enzyme-linked immunosorbent assay (bELISA) kit (LSI, Lissieu, France) previously validated for the testing of reindeer serum samples for CvHV2 specific antibodies . Positive and negative controls for cattle provided in the bELISA kit were included on each plate.

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A serological screening was also carried out for the detection of antibodies against pestivirus using a commercial bELISA kit for the detection of BVDV antibodies in cattle (SERELISA™ BVD p80 Ab Mono blocking; Synbiotics Europe SAS, Lyon, France), which has been previously evaluated for testing reindeer serum samples .

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DNA was extracted from swab samples with a QIAamp DNA MiniKit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Different subsets of reindeer samples were analyzed for the presence of DNA specific to CvHV2 (n = 200), Chlamydiaceae (n = 135) or M. conjunctivae (n = 197) depending on the availability of DNA extracted from the swab samples.

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A nested pan-alphaherpesvirus PCR targeting the UL27 gene encoding glycoprotein B (gB) of CvHV2 was performed as described by Ros and Bèlak for CvHV2 (strain Salla82, Finland; Ek-Kommonen et al. , initially named rangiferine herpesvirus 1), in a subset of samples (n = 200). DNA extracted from purified CvHV2 was used as positive control and diethylpryocarbonate (DEPC) water was used as negative control.

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One hundred and thirty-five (n = 135) samples were analyzed (National Veterinary Institute, Sweden) by a TaqMan real-time PCR specific for members of the family Chlamydiaceae, targeting the 23S rRNA operon . The cut-off value was set at Ct > 38, so every sample with a threshold cycle (Ct) below that was considered positive for the presence of Chlamydiaceae DNA.

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A specific PCR assay based on unique sequences of the LppS gene was used for detection of Mycoplasma spp. in a subset of samples (n = 197). DNA purified from M. conjunctivae strain HRC/581 originating from sheep (ATCC 25834; NCTC 10147) was used as positive control , while DEPC water was used as negative control.

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Amplified DNA products were separated by agarose gel (1.0%) electrophoresis and stained with ethidium bromide. Amplicons similar to the expected size (139 bp for M. conjunctivae and 294 bp for CvHV2) were purified and sequenced. Consensus amplicon sequences were assembled with the Chromas pro software (version 1.7.7, Technelysium Pty Ltd., South Brisbane, QLD, Australia) and blasted in GenBank (NCBI, USA) for confirmation and comparison to available matching sequences of CvHV2 or M. conjunctivae.

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Swab samples from the conjunctiva of the most affected eye in animals with clinical signs of IKC, and one of the eyes when sampling apparently healthy animals were cultivated on two 5.0% sheep blood agar plates, incubated aerobically and anaerobically, and on one lactose-saccharose-bromothymol blue agar plate (equal to MacConkey agar) incubated aerobically. All plates were incubated at 37 °C and inspected after 24 and 48 h. Bacterial growth was categorized as rich, moderate, or poor. Dominant colonies or colonies suspected as relevant, were subcultured for purity and characterized (morphology, Gram staining, catalase, oxidase). API® strips (bioMérieux, Marcy l’Etoile, France) were used for bacterial identification. If identification to species level was not successful, isolates were characterized by 16S ribosomal RNA (16S rRNA) gene sequencing. The proximal part of the 16S rRNA gene was amplified and sequenced using the MicroSeq® 500 16S rRNA Bacterial Identification Kits (Applied Biosystems, Foster City, CA, USA). The sequencing reactions were run on a capillary sequencer 3130xl Genetic Analyzer (Applied Biosystems Life Technologies, Thermo Fisher Scientific Inc., Waltham, MA). Sequences were analyzed using the CLC bio Combined Workbench (CLC bio, QIAGEN, Aarhus, Denmark) or the BioEdit program (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and homology search was performed using NCBI GeneBlast2 program.

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Seroprevalence of antibodies against alphaherpesvirus was 79.5% (31/39) in adults and 24% (31/129) in calves. For calves, the seroprevalence increased with increasing severity of clinical signs, while for adults, no such difference was present and the seroprevalence was 66.7–100% regardless of the clinical score (Figs. 2 & 4).Fig. 2Alphaherpesvirus seroprevalence among reindeer without eye lesions (0), with mild (1), or moderate/severe (2) eye lesions presented as seronegative (blue) and seropositive (red) comparing calves (left) and adults (right)

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There was a statistically significant and positive association between the presence of clinical signs of IKC and the presence of antibodies against alphaherpesvirus in calves (p = 0.006). No association was found for adults (p = 0.682) or for the whole sample set when age class was not specified (p = 0.143).

other

Eight out of 211 animals (3.8%) were seropositive for the presence of antibodies against gammaherpesvirus. Among animals with known clinical status (n = 140), it was not possible to establish an association between the presence of antibodies against gammaherpesvirus and the presence of clinical signs of IKC (p = 0.257), a lack of association that persisted also when addressing calves (p = 0.394) or adults (p = 0.312) separately.

other

The presence of antibodies against pestivirus could not be associated with the presence of clinical signs of IKC, either when analyzing calves and adults together (n = 140; p = 0.761), or among just calves (p = 1.000). Since all seropositive animals were calves no such calculation was possible for adults as a separate category. If animals without known clinical information were included (n = 211), 15 reindeer had antibodies against pestivirus (7.1%). Six additional animals were classified as doubtful in the ELISA, retested with the same results, and subsequently excluded from our statistical analysis.

review

In total, 202 animals were screened for the presence of antibodies against all the three viruses (Table 2). Among them, ten animals had antibodies against pestivirus and alphaherpesvirus, while four had antibodies against gamma- and alphaherpesvirus. None of the animals were seropositive for both pestivirus and gammaherpesvirus, or for all the three viruses. Among the pestivirus seropositive animals, 66.7% (10/15) were also seropositive to alphaherpesvirus, with a statistical significant association between the presence of antibodies against alphaherpesvirus and pestivirus (p = 0.003). This association did not exist in any of the other combinations of antibodies.Table 2Semi-domesticated reindeer screened by ELISA (n = 202) for antibodies against alphaherpes-, gammaherpes- and pestivirusOcular clinical signs a012Not registeredAlphaherpesvirus29 / 89 (32.6)7 / 24 (29.2)16 / 27 (59.3)10 / 62 (16.1)Gammaherpesvirus3 / 89 (3.4)2 / 24 (8.3)2 / 27 (7.4)1 / 62 (1.6)Pestivirus7 / 86b (8.1)4 / 24 (16.7)1 / 27 (3.7)3 / 59b (5.1)Gammaherpesvirus and Alphaherpesvirus1 / 31 / 22 / 20 / 1Pestivirus and Alphaherpesvirus6 / 74 / 40 / 10 / 3Results are presented as positive/screened (%). No individuals were seropositive for both gammaherpes- and pestivirus, nor for the combination of all the three virusesaThe severity of the ocular disease was scored with 0 for asymptomatic animals, 1 for animals with increased lacrimation and/or mild conjunctivitis or 2 for animals with moderate to severe clinical signs of IKCbThree samples were considered doubtful and run twice with the same result and therefore excluded from the analysis

clinical case

The CvHV2 PCR results showed a positive significant association between the presence of CvHV2 DNA in the conjunctival swab samples and the severity of the clinical signs of IKC. This association was valid both for the total number of animals independently of age-class (p < 0.001) and also for calves (p = 0.002) and adults (p = 0.022) as separate groups (Figs. 3 & 4). Nine out of 59 of the asymptomatic animals (15.3%) had CvHV2 DNA in their eyes.Fig. 3Alphaherpesvirus PCR results (eye swab) among reindeer without eye lesions (0), with mild (1), or moderate/severe (2) eye lesions presented as PCR negative (blue) and PCR positive (red) comparing calves (left) and adults (right)Fig. 4Alphaherpesvirus seroprevalence (a) and alphaherpesvirus PCR results (eye swab) (b) among reindeer without eye lesions (0), with mild (1), or moderate/severe (2) eye lesions presented as percentage comparing calves (left) and adults (right)

study

DNA specific for Chlamydiacea was detected in 16 of 135 animals (11.9%), eight with and eight without clinical signs of IKC. No statistical association could be established (p = 1.000) between the presence of clinical signs of IKC and the detection of bacteria from the family Chlamydiacea.

other

A total of 202 animals, one eye from each, were sampled for bacteriology. Bacteria were cultivated from the conjunctiva of 152 reindeer, of which 35 presented clinical signs of IKC, 52 did not show signs of IKC, and the clinical status was unknown for 65 individuals. In animals from which clinical information was not available, bacteria such as Acinetobacter spp. (n = 13), Aeromonas hydrophila (n = 5), Escherichia coli (n = 9), Micrococcus lylae (n = 8) and Staphylococcus spp. (n = 3) were the major findings.

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One of the M. bovoculi isolates was cultured from the eye swab from one reindeer with mild signs of IKC in Ifjordfjellet (Finmmark County, Norway) (Fig. 1). All other Moraxella spp. isolates were obtained from two herds with active IKC outbreaks. However, no significant differences were found between the presence of Moraxella spp. in reindeer with IKC and reindeer without IKC (p = 0.148).

other

Several species of bacteria were isolated from the eyes of symptomatic and non-symptomatic animals (Fig. 5), but statistical association between the presence of any of these bacteria and clinical signs of IKC could not be established. For 52 of the reindeer (25.7%, one conjunctival swab from each animal obtained for bacteriology), the cultivation results were not conclusive due to poor bacterial growth or the absence of a dominant species, i.e. nonspecific mixed culture.Fig. 5Bacterial species that dominated the culture plates inoculated with swab samples from eyes of semi-domesticated Eurasian reindeer (Rangifer tarandus tarandus) without (a) and with (b) clinical signs of infectious keratoconjunctivitis (IKC), including the percentage of swabs from which the bacteria were isolated

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Bacterial species that dominated the culture plates inoculated with swab samples from eyes of semi-domesticated Eurasian reindeer (Rangifer tarandus tarandus) without (a) and with (b) clinical signs of infectious keratoconjunctivitis (IKC), including the percentage of swabs from which the bacteria were isolated

study

There was a positive correlation between the presence of antibodies against alphaherpesvirus and the presence of clinical signs of IKC in calves (p < 0.001), but not in adults (p = 0.417). Calves had higher scores of IKC clinical signs and seemed to be suffering more acute forms of the disease as compared to older animals, most likely due to the lack of previous exposures to the virus and therefore absence of immunity during the establishment of the infection. The higher seroprevalence of antibodies against alphaherpesvirus in adults as compared to calves was expected, since alphaherpesviruses produce lifelong infections , which is also in line with previous findings .

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Adult animals that were previously exposed and infected could be carrying a latent infection without showing clinical signs at the time of sampling. Reactivation from this latency stage will stimulate a quick onset of the immune response protecting the animals from developing disease.

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The detection of CvHV2 DNA in eye swabs by PCR and the prevalence of PCR positive individuals, which increased with the severity of the clinical signs of IKC (Fig. 3), supports the hypothesis that CvHV2 is a primary agent in the development of IKC in reindeer . CvHV2 DNA was also amplified by PCR from the eyes of 15.3% of the apparently healthy reindeer. These asymptomatic animals may have been sampled during the incubation period of the disease or the virus may have been replicating in the conjunctival mucosa without producing clinical IKC signs.

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It was not possible to detect CvHV2 DNA from 12 animals with mild and one with severe signs of IKC. The signs included in the first category are defined as nonspecific signs associated to IKC, such as increased lacrimation and mild conjunctivitis, which can have a non-infectious cause such as dust, dirt, allergies or minor trauma. In a natural outbreak of IKC in reindeer, the virus was most often detected and isolated at higher titers, from animals with mild or early stage signs of IKC and only from a few animals with severe signs. It was hypothesized that the peak of viral replication and shedding occurred at earlier stages, whereas secondary bacterial infections were dominant in later stages of the disease [15, 21]. It is also possible that the virus, during the late stages of IKC, is entering the latency stage, shutting down the active replication cycle and the shedding of progeny virus.

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The statistical analysis of our data supports these observations. There was a strong association between the presence of CvHV2 DNA in the eye of the animals and the presence of clinical signs (p < 0.001). These results indicate that during an acute CvHV2 infection, with active replication of the virus in the conjunctival mucosa, clinical signs characteristic of IKC may develop.

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Interestingly, all CvHV2 seropositive calves were also positive for the presence of CvHV2 DNA in their eyes (12/12). The ratio of PCR-positive animals also positive for the presence of antibodies against CvHV2 increased with the development of more severe clinical signs of IKC (Score 0 = 0% (0/5), Score 1 = 16,7% (1/6), Score 2 = 64,7% (11/17)). Those results support the hypothesis of CvHV2 infected calves seroconverting in later stages of the disease. Seronegative calves with positive PCR results (n = 16) might still be in the window period of the disease, in which the animals have been infected, but it is not possible to detect antibodies.

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Despite serological screenings reporting antibodies against both pestivirus and gammaherpesvirus in reindeer in Fennoscandia [22, 27], little information is available about their pathogenic potential related to eye disease. The lack of association between the presence of antibodies against pestivirus and gammaherpesvirus and the presence of clinical signs of IKC indicated that these viruses do not contribute to the development of IKC in reindeer. However, it has been speculated that there might be an interaction between pestivirus and alphaherpesvirus in reindeer, in the way that infection with one of the viruses increases the chances of infection by the second virus . The statistical association between the presence of antibodies against pestivirus and alphaherpesvirus in our study could reinforce this hypothesis (p = 0.003). Interestingly, all animals that were seropositive to both alphaherpes- and pestivirus (n = 10) were sampled from the same herd and period, which could also means that the animals in that herd were managed differently, increasing the possibility of exposure to the pestivirus.

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Bacteria belonging to the genus Moraxella were isolated from the eyes of eleven animals with, and five animals without clinical signs of IKC. Seven of these isolates were characterized as M. bovoculi. Together with M. bovis, M. bovoculi has been identified as a causative agent of IBK in cattle [47, 48], but a randomized blinded challenge study suggested that M. bovoculi was not causally related with IBK in this species .

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A statistically significant association did not exist between the isolation of Moraxella spp. and the presence clinical signs of IKC in reindeer (p = 0.461). However, fifteen of the samples from which Moraxella spp. were cultivated were obtained during two different IKC outbreaks, in Sørreisa (Norway) and Karesuando (Sweden), supporting the hypothesis of a biological significance of Moraxella spp. in the development of IKC in reindeer.

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Dickey et al. showed that there are large genomic differences between Moraxella bovoculi isolates from IBK affected cattle and asymptomatic animals. These results open the possibility to the presence of different strains of Moraxella bovoculi with different pathogenic potential. Therefore, further genomic investigations should be carried out in order to identify if pathogenic characteristics are present in the Moraxella spp. isolated from the eyes of apparently healthy and diseased semi-domesticated reindeer.

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DNA specific for Chlamydiacea and M. conjunctivae, both known to be involved in IKC and eye infections in other host species, was detected in a few reindeer samples, both from animals with or without clinical signs of IKC. However, the lack of association between the presence of Chlamydiacea or M. conjunctivae DNA and the presence of clinical signs of IKC may suggest that these bacteria are not essential to the pathogenesis of IKC in reindeer.

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A great variety of other bacteria were isolated from the eyes of both healthy and IKC affected animals (Fig. 5), including some potentially pathogenic bacteria (e.g. Pseudomonas spp. or Staphylococcus spp. [51, 52]), but also several bacterial species that do not have any known importance in veterinary medicine, most likely representing contamination from the environment (i.e. dust, soil, feces etc.). However, no significant association could be identified between the presence of any of the isolated bacteria and the presence of clinical signs of IKC, which may suggest that none of these bacteria have a significant importance for the development of IKC in reindeer. Bacteria did not grow in 24.8% of the cultured plates, a percentage that seems higher than those reported in other ruminant species [13, 14]. This increment could be attributed to the somewhat long (2–9 days) transport time for some samples to the laboratory which may in some cases have reduced the survival of bacteria in the samples prior to cultivation, or simply reflect a different environment (i.e. free ranging animals as opposed to livestock).

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Some species of bacteria might require the presence of damage of the mucosal membrane to be able to establish an infection, as occurs with the cytopathic effect (CPE) produced by CvHV2 during its lytic cycle. A depression of the cell-mediated immunity due to virus-induced lymphocytolysis after infection with BoHV1 has been described , and it is relevant to think that CvHV2, a close relative to BoHV1, could produce a similar effect on the reindeer’s immune system, favoring the establishment of secondary bacterial infections. On the other hand, the possible role of bacterial infection as a trigger for the reactivation of latent CvHV2 cannot be discarded either. Bovine Herpesvirus 4 (BoHV4) can enter a lytic replication cycle from latency after endometrial infection with E. coli, causing uterine disease in cattle . However, BoHV4 belongs to the subfamily Gammaherpesvirinae, and whether this finding can be applied to CvHV2 or any other ruminant alphaherpesviruses remains unclear and should be studied in further detail.

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Among the microorganism identified in this study, CvHV2 is the most plausible candidate as the causative agent of IKC in semi-domesticated reindeer, and pestivirus and gammaherpesvirus may be discarded as primary causative agents of IKC in reindeer. The isolation of M. bovoculi during two different outbreaks of IKC makes this bacterial species an alternative candidate as a possible primary agent of this disease, or maybe more likely, as a secondary and opportunistic pathogen, following a CvHV2 infection as is probably the case for other bacterial species identified in this study. Further studies should be carried out to better understand the infection biology and the pathogenesis of IKC in reindeer.

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IgA nephropathy (IgAN) is the most common primary glomerulonephritis and characterized by various degrees of intrinsic cell proliferation, especially mesangial cells (MCs), in the affected glomerulus, and mononuclear leukocyte infiltration in the glomerulus and renal interstitium1234, with insidious progression to end-stage renal disease in up to 50% of the patients56. Deposition of predominantly glomerular IgA immune complexes (ICs) or IgA immune aggregates167, and activation of innate immunity, followed by T cell activation and resultant inflammatory responses have been considered as a leading cause of the disease378. However, the exact pathogenic mechanism underlying IgAN remains largely unknown, and specific treatment for the renal disease is still insufficient. NLRP3 inflammasome links innate and adaptive immunity and is involved in secretion of IL-1β and IL-18 by various immune cells9101112 in an inflammatory condition13141516. Innate immunity is triggered by endogenous or environmental insults through assembly of the NLRP3 inflammasome1012, which has been observed in many cell types, such as macrophages, dendritic cells (DCs), T cells, and some epithelial cells101112. Recently, several studies have suggested that NLRP3 inflammasome may be implicated in the pathogenesis of IgAN in pateints161718. However, the causal-relationship between the inflammasome and the pathogenesis of IgAN is yet to be determined.

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We have previously showed that IL-1β and related molecular mechanisms are involved in the pathogenesis of a spontaneously occurring IgAN model in ddY mice19, and protein levels of NLRP3 and IL-1β, and caspase-1 activity in the kidney are significantly increased in a passively induced mouse IgAN model, but this effect is inhibited by potent chemical inhibitors that are originally derived from traditional Chinese medicines2021, suggesting that NLRP3 inflammasome may play a role in the pathogenesis of IgAN.

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Herein, we hypothesized that activation of NLRP3 inflammasome contributes to the pathogenesis of IgAN and targeting the inflammasome may be beneficial for the treatment of the renal disease. This is the first report that addresses the NLRP3 inflammasome activation specifically involved in the development and progression of IgAN, based on our mechanistic investigations using cultured macrophages, DCs, glomerular MCs, and renal tubular epithelial cells (TECs), NLRP3 knockout (NLRP3 KO) mice, and gene delivery of shRNA using a kidney-targeting, ultrasound-mediated microbubble technique.

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First, we examined whether IgA ICs can activate NLRP3 inflammasome in macrophages and DCs. As shown in Fig. 1, IgA ICs increased the protein levels of NLRP3 and pro-IL-1β in J774A.1 macrophages (Fig. 1a). IgA ICs induced caspase-1 activation in the presence of ATP evidenced by the increased active caspase-1 in the supernatants of J774A.1 cells (Fig. 1b). Also, IgA ICs induced IL-1β secretion in the presence of ATP as evidenced by increased levels of mature IL-1β in the supernatants detected by Western blot analysis and ELISA (Fig. 1c). As shown in Fig. 1d, IgA ICs induced IL-1β secretion was dependent on NLRP3 inflammasome, as the level of IL-1β secretion was significantly reduced in shRNA targeting NLRP3 macrophages (Fig. 1d). The role of NLRP3 in IgA ICs-induced IL-1β secretion was confirmed in peritoneal macrophages, as caspase-1 activation and IL-1β secretion were significantly reduced in the cells derived from NLRP3 KO mice compared to those of peritoneal macrophages from WT mice (Fig. 1d). In addition, a non-canonical pathway is implicated in the activation of the NLRP3 inflammasome, which, in mice, requires caspase-11 (while caspase-4 and caspase-5 in humans)222324. As shown in Fig. 1e, IgA ICs activated shSC (shscramble) macrophages showed significantly higher caspase-11 mRNA levels than saline control, and these effects were either markedly inhibited or absent in J774A.1 macrophages stable transfection with shRNA targeting shcaspase-11. Furthermore, the levels of IL-1β were significantly lower in the shcaspase-11 cells than in the shSC cells (Fig. 1f).

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In addition, IgA ICs increased TNF-α secretion, percentages of CD11c+CD40+ and CD11c+CD86+ (Fig. 2a), and IL-1β secretion (Fig. 2b) in bone marrow derived DCs (BMDCs) from WT mice, but this effect was significantly inhibited in BMDCs from NLRP3 KO mice. Using an OVA-specific T cell proliferation assay, IgA ICs-primed BMDCs from WT mice induced proliferation of CD4+ T cells and their secretion of IFN-γ, IL-17A and IL-4 (Fig. 2c), and, except IL-4, these effects were greatly inhibited in BMDCs from NLRP3 KO mice. These results suggest that NLRP3 inflammasome plays an important role in IgA ICs-mediated IL-1β secretion and T cell activation.

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