Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Addendum: Immune clearance of highly pathogenic SIV infection.

This corrects the article DOI: 10.1038/nature12519.

Terumo spectra optia leukapheresis of cynomolgus macaques for hematopoietic stem cell and T cell collection.

Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen-specific lymphocytes for in-depth immunological analysis, and latently-infected CD4+ T-cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T-cells from cynomolgus macaques as small as 3 kg using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 × 109 total nucleated cells from mobilized animals and 1.2 × 109 total nucleated cells from nonmobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.

Vaccine-Mediated Inhibition of the Transporter Associated with Antigen Processing Is Insufficient To Induce Major Histocompatibility Complex E-Restricted CD8+ T Cells in Nonhuman Primates.

Major histocompatibility complex E (MHC-E) is a highly conserved nonclassical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs coexpressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus 2 ICP47, bovine herpes virus 1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia but not MHC-E. These data demonstrate that, in contrast to results in mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.IMPORTANCE Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.

Viral opportunistic infections in Mauritian cynomolgus macaques undergoing allogeneic stem cell transplantation mirror human transplant infectious disease complications.

Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.

The Role of MHC-E in T Cell Immunity Is Conserved among Humans, Rhesus Macaques, and Cynomolgus Macaques.

MHC-E is a highly conserved nonclassical MHC class Ib molecule that predominantly binds and presents MHC class Ia leader sequence-derived peptides for NK cell regulation. However, MHC-E also binds pathogen-derived peptide Ags for presentation to CD8+ T cells. Given this role in adaptive immunity and its highly monomorphic nature in the human population, HLA-E is an attractive target for novel vaccine and immunotherapeutic modalities. Development of HLA-E-targeted therapies will require a physiologically relevant animal model that recapitulates HLA-E-restricted T cell biology. In this study, we investigated MHC-E immunobiology in two common nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM). Compared to humans and MCM, RM expressed a greater number of MHC-E alleles at both the population and individual level. Despite this difference, human, RM, and MCM MHC-E molecules were expressed at similar levels across immune cell subsets, equivalently upregulated by viral pathogens, and bound and presented identical peptides to CD8+ T cells. Indeed, SIV-specific, Mamu-E-restricted CD8+ T cells from RM recognized antigenic peptides presented by all MHC-E molecules tested, including cross-species recognition of human and MCM SIV-infected CD4+ T cells. Thus, MHC-E is functionally conserved among humans, RM, and MCM, and both RM and MCM represent physiologically relevant animal models of HLA-E-restricted T cell immunobiology.

Combination Adenovirus and Protein Vaccines Prevent Infection or Reduce Viral Burden after Heterologous Clade C Simian-Human Immunodeficiency Virus Mucosal Challenge.

HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of adenovirus (Ad) vectors expressing HIV 1086.C (clade C) envelope glycoprotein (Env), SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with nonreplicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups, plasma and buccal Env-specific IgG, tier 1 heterologous neutralizing antibodies, and antibody-dependent cell-mediated viral inhibition were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. After three intrarectal exposures to heterologous tier 1 clade C SHIV, all 10 sham-vaccinated controls were infected, whereas 4/10 SAd7- and 3/10 Ad4-vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared to controls; the lowest plasma viral burden was found in animals vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus, recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines.IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV vaccine platforms, there have not been any direct comparisons of the replicating to the nonreplicating format. The present study directly compared the replicating SAd7 to nonreplicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, the time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.

Correction: Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues.

[This corrects the article DOI: 10.1371/journal.ppat.1006219.].

Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues.

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.

Immune clearance of highly pathogenic SIV infection.

Established infections with the human and simian immunodeficiency viruses (HIV and SIV, respectively) are thought to be permanent with even the most effective immune responses and antiretroviral therapies only able to control, but not clear, these infections. Whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of HIV-infected individuals. We recently reported that approximately 50% of rhesus macaques (RM; Macaca mulatta) vaccinated with SIV protein-expressing rhesus cytomegalovirus (RhCMV/SIV) vectors manifest durable, aviraemic control of infection with the highly pathogenic strain SIVmac239 (ref. 5). Here we show that regardless of the route of challenge, RhCMV/SIV vector-elicited immune responses control SIVmac239 after demonstrable lymphatic and haematogenous viral dissemination, and that replication-competent SIV persists in several sites for weeks to months. Over time, however, protected RM lost signs of SIV infection, showing a consistent lack of measurable plasma- or tissue-associated virus using ultrasensitive assays, and a loss of T-cell reactivity to SIV determinants not in the vaccine. Extensive ultrasensitive quantitative PCR and quantitative PCR with reverse transcription analyses of tissues from RhCMV/SIV vector-protected RM necropsied 69-172 weeks after challenge did not detect SIV RNA or DNA sequences above background levels, and replication-competent SIV was not detected in these RM by extensive co-culture analysis of tissues or by adoptive transfer of 60 million haematolymphoid cells to naive RM. These data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory T-cell-mediated immune surveillance elicited and maintained by cytomegalovirus vectors.

Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus.

The natural killer cell receptor NKG2D activates NK cells by engaging one of several ligands (NKG2DLs) belonging to either the MIC or ULBP families. Human cytomegalovirus (HCMV) UL16 and UL142 counteract this activation by retaining NKG2DLs and US18 and US20 act via lysomal degradation but the importance of NK cell evasion for infection is unknown. Since NKG2DLs are highly conserved in rhesus macaques, we characterized how NKG2DL interception by rhesus cytomegalovirus (RhCMV) impacts infection in vivo. Interestingly, RhCMV lacks homologs of UL16 and UL142 but instead employs Rh159, the homolog of UL148, to prevent NKG2DL surface expression. Rh159 resides in the endoplasmic reticulum and retains several NKG2DLs whereas UL148 does not interfere with NKG2DL expression. Deletion of Rh159 releases human and rhesus MIC proteins, but not ULBPs, from retention while increasing NK cell stimulation by infected cells. Importantly, RhCMV lacking Rh159 cannot infect CMV-naïve animals unless CD8+ cells, including NK cells, are depleted. However, infection can be rescued by replacing Rh159 with HCMV UL16 suggesting that Rh159 and UL16 perform similar functions in vivo. We therefore conclude that cytomegaloviral interference with NK cell activation is essential to establish but not to maintain chronic infection.

Cross-Species Rhesus Cytomegalovirus Infection of Cynomolgus Macaques.

Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68-1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68-1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68-1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68-1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68-1 and 68-1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics.

Effect of Anti-IL-15 Administration on T Cell and NK Cell Homeostasis in Rhesus Macaques.

IL-15 has been implicated as a key regulator of T and NK cell homeostasis in multiple systems; however, its specific role in maintaining peripheral T and NK cell populations relative to other γ-chain (γc) cytokines has not been fully defined in primates. In this article, we address this question by determining the effect of IL-15 inhibition with a rhesusized anti-IL-15 mAb on T and NK cell dynamics in rhesus macaques. Strikingly, anti-IL-15 treatment resulted in rapid depletion of NK cells and both CD4(+) and CD8(+) effector memory T cells (TEM) in blood and tissues, with little to no effect on naive or central memory T cells. Importantly, whereas depletion of NK cells was nearly complete and maintained as long as anti-IL-15 treatment was given, TEM depletion was countered by the onset of massive TEM proliferation, which almost completely restored circulating TEM numbers. Tissue TEM, however, remained significantly reduced, and most TEM maintained very high turnover throughout anti-IL-15 treatment. In the presence of IL-15 inhibition, TEM became increasingly more sensitive to IL-7 stimulation in vivo, and transcriptional analysis of TEM in IL-15-inhibited monkeys revealed engagement of the JAK/STAT signaling pathway, suggesting alternative γc cytokine signaling may support TEM homeostasis in the absence of IL-15. Thus, IL-15 plays a major role in peripheral maintenance of NK cells and TEM However, whereas most NK cell populations collapse in the absence of IL-15, TEM can be maintained in the face of IL-15 inhibition by the activity of other homeostatic regulators, most likely IL-7.

Effect of IL-7 Therapy on Naive and Memory T Cell Homeostasis in Aged Rhesus Macaques.

Aging is associated with gradual deterioration of adaptive immune function, a hallmark of which is the profound loss of naive T cells (TN) associated with decline in thymic output and export of new cells into the peripheral T cell pool. Because the lymphotropic cytokine IL-7 plays crucial roles in both development of TN in the thymus and TN homeostasis in the periphery, we sought to determine the extent to which therapeutic administration of IL-7 could reverse TN deficiency in aging rhesus macaques (RM), either by enhancement of the demonstrably reduced thymopoiesis or by peripheral TN expansion. Our results indicate that treatment of both adult (8-15 y) and old (>20 y) RM with recombinant simian IL-7 (rsIL-7) results in only transient increases in peripheral CD4(+) and CD8(+) TN numbers with no long-term benefit, even with repeated therapy. This transient effect was due to peripheral TN expansion and not enhanced thymic function, and appeared to be limited by induction of IL-7 nonresponsiveness. However, rsIL-7 therapy had a more promising effect on the central memory T cell (TCM) population (both CD4(+) and CD8(+)) in adult and old RM, doubling the numbers of these cells in circulation and maintaining this larger population long term. IL-7 therapy did not reduce TCR diversity of the memory T cell compartment, suggesting that rsIL-7-induced expansion was symmetrical. Thus, although rsIL-7 failed to counter age-associated TN loss, the ability of this therapy to expand clonotypically diverse CD4(+) and CD8(+) TCM populations might potentially improve adaptive immune responsiveness in the elderly.

Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine.

The acquired immunodeficiency syndrome (AIDS)-causing lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) effectively evade host immunity and, once established, infections with these viruses are only rarely controlled by immunological mechanisms. However, the initial establishment of infection in the first few days after mucosal exposure, before viral dissemination and massive replication, may be more vulnerable to immune control. Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell (T(EM)) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic SIV(MAC239) infection early after mucosal challenge. Thirteen of twenty-four rhesus macaques receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (versus 0 of 9 DNA/Ad5-vaccinated rhesus macaques) manifested early complete control of SIV (undetectable plasma virus), and in twelve of these thirteen animals we observed long-term (≥1 year) protection. This was characterized by: occasional blips of plasma viraemia that ultimately waned; predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; no depletion of effector-site CD4(+) memory T cells; no induction or boosting of SIV Env-specific antibodies; and induction and then loss of T-cell responses to an SIV protein (Vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8(+) T-cell responses in the vaccine phase, and occurred without anamnestic T-cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8(+) or CD4(+) lymphocyte depletion and, at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations that indicate the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated T(EM) responses might significantly contribute to an efficacious HIV/AIDS vaccine.

T cell inactivation by poxviral B22 family proteins increases viral virulence.

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.

Neisseria infection of rhesus macaques as a model to study colonization, transmission, persistence, and horizontal gene transfer.

The strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe-host interactions. We developed a rhesus macaque model for studying Neisseria-host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria, as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.

Chikungunya viruses that escape monoclonal antibody therapy are clinically attenuated, stable, and not purified in mosquitoes.

UNLABELLED: Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1(-/-) mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle. IMPORTANCE: Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.

Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection.

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4(+) CCR5(+) effector-memory T (T(EM)) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4(+) memory T cell proliferation appears to prevent collapse of effector site CD4(+) T(EM) cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4(+) T(EM) cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4(+) T(EM) cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4(+) T(EM) cells from central-memory T (T(CM)) cell precursors. The instability of effector site CD4(+) T(EM) cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5(-) CD4(+) T(CM) cells. These data suggest that although CD4(+) T(EM) cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4(+) T(CM) cells.

CD4 T cell immunity is critical for the control of simian varicella virus infection in a nonhuman primate model of VZV infection.

Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses.