Mayaro virus pathogenesis and immunity in rhesus macaques.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

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.

Infection with chikungunya virus confers heterotypic cross-neutralizing antibodies and memory B-cells against other arthritogenic alphaviruses predominantly through the B domain of the E2 glycoprotein.

Infections with Chikungunya virus, a mosquito-borne alphavirus, cause an acute febrile syndrome often followed by chronic arthritis that persists for months to years post-infection. Neutralizing antibodies are the primary immune correlate of protection elicited by infection, and the major goal of vaccinations in development. Using convalescent blood samples collected from both endemic and non-endemic human subjects at multiple timepoints following suspected or confirmed chikungunya infection, we identified antibodies with broad neutralizing properties against other alphaviruses within the Semliki Forest complex. Cross-neutralization generally did not extend to the Venezuelan Equine Encephalitis virus (VEEV) complex, although some subjects had low levels of VEEV-neutralizing antibodies. This suggests that broadly neutralizing antibodies elicited following natural infection are largely complex restricted. In addition to serology, we also performed memory B-cell analysis, finding chikungunya-specific memory B-cells in all subjects in this study as remotely as 24 years post-infection. We functionally assessed the ability of memory B-cell derived antibodies to bind to chikungunya virus, and related Mayaro virus, as well as the highly conserved B domain of the E2 glycoprotein thought to contribute to cross-reactivity between related Old-World alphaviruses. To specifically assess the role of the E2 B domain in cross-neutralization, we depleted Mayaro and Chikungunya virus E2 B domain specific antibodies from convalescent sera, finding E2B depletion significantly decreases Mayaro virus specific cross-neutralizing antibody titers with no significant effect on chikungunya virus neutralization, indicating that the E2 B domain is a key target of cross-neutralizing and potentially cross-protective neutralizing antibodies.

Development of a next-generation chikungunya virus vaccine based on the HydroVax platform.

Chikungunya virus (CHIKV) is an emerging/re-emerging mosquito-borne pathogen responsible for explosive epidemics of febrile illness characterized by debilitating polyarthralgia and the risk of lethal infection among the most severe cases. Despite the public health risk posed by CHIKV, no vaccine is currently available. Using a site-directed hydrogen peroxide-based inactivation approach, we developed a new CHIKV vaccine, HydroVax-CHIKV. This vaccine technology was compared to other common virus inactivation approaches including ß-propiolactone (BPL), formaldehyde, heat, and ultraviolet (UV) irradiation. Heat, UV, and BPL were efficient at inactivating CHIKV-181/25 but caused substantial damage to neutralizing epitopes and failed to induce high-titer neutralizing antibodies in vaccinated mice. HydroVax-CHIKV and formaldehyde-inactivated CHIKV retained intact neutralizing epitopes similar to live virus controls but the HydroVax-CHIKV approach demonstrated a more rapid rate of virus inactivation. HydroVax-CHIKV vaccination induced high neutralizing responses to homologous and heterologous CHIKV clades as well as to other alphaviruses including Mayaro virus, O'nyong'nyong virus, and Una virus. Following heterologous infection with CHIKV-SL15649, HydroVax-CHIKV-immunized mice were protected against viremia, CHIKV-associated arthritic disease, and lethal CHIKV infection by an antibody-dependent mechanism. In contrast, animals vaccinated with Heat- or UV-inactivated virus showed no protection against viremia in addition to demonstrating significantly exacerbated CD4+ T cell-mediated footpad swelling after CHIKV infection. Together, these results demonstrate the risks associated with using suboptimal inactivation methods that fail to elicit protective neutralizing antibody responses and show that HydroVax-CHIKV represents a promising new vaccine candidate for prevention of CHIKV-associated disease.

Suppression of human and simian immunodeficiency virus replication with the CCR5-specific antibody Leronlimab in two species.

The CCR5-specific antibody Leronlimab is being investigated as a novel immunotherapy that can suppress HIV replication with minimal side effects. Here we studied the virological and immunological consequences of Leronlimab in chronically CCR5-tropic HIV-1 infected humans (n = 5) on suppressive antiretroviral therapy (ART) and in ART-naïve acutely CCR5-tropic SHIV infected rhesus macaques (n = 4). All five human participants transitioned from daily combination ART to self-administered weekly subcutaneous (SC) injections of 350 mg or 700 mg Leronlimab and to date all participants have sustained virologic suppression for over seven years. In all participants, Leronlimab fully occupied CCR5 receptors on peripheral blood CD4+ T cells and monocytes. In ART-naïve rhesus macaques acutely infected with CCR5-tropic SHIV, weekly SC injections of 50 mg/kg Leronlimab fully suppressed plasma viremia in half of the macaques. CCR5 receptor occupancy by Leronlimab occurred concomitant with rebound of CD4+ CCR5+ T-cells in peripheral blood, and full CCR5 receptor occupancy was found in multiple anatomical compartments. Our results demonstrate that weekly, self-administered Leronlimab was safe, well-tolerated, and efficacious for long-term virologic suppression and should be included in the arsenal of safe, easily administered, longer-acting antiretroviral treatments for people living with HIV-1. Trial Registration: ClinicalTrials.gov Identifiers: NCT02175680 and NCT02355184.

Antibody-based CCR5 blockade protects Macaques from mucosal SHIV transmission.

In the absence of a prophylactic vaccine, the use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition by uninfected individuals is a promising approach to slowing the epidemic, but its efficacy is hampered by incomplete patient adherence and ART-resistant variants. Here, we report that competitive inhibition of HIV Env-CCR5 binding via the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of CCR5-tropic SHIVSF162P3. Injection of Leronlimab weekly at 10 mg/kg provides significant but partial protection, while biweekly 50 mg/kg provides complete protection from SHIV acquisition. Tissue biopsies from protected macaques post challenge show complete CCR5 receptor occupancy and an absence of viral nucleic acids. After Leronlimab washout, protected macaques remain aviremic, and adoptive transfer of hematologic cells into naïve macaques does not transmit viral infection. These data identify CCR5 blockade with Leronlimab as a promising approach to HIV prophylaxis and support initiation of clinical trials.

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.

The human IL-15 superagonist N-803 promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-suppressed, SHIV-infected macaques.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.

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.