Rapid Inflammasome Activation following Mucosal SIV Infection of Rhesus Monkeys.

The earliest events following mucosal HIV-1 infection, prior to measurable viremia, remain poorly understood. Here, by detailed necropsy studies, we show that the virus can rapidly disseminate following mucosal SIV infection of rhesus monkeys and trigger components of the inflammasome, both at the site of inoculation and at early sites of distal virus spread. By 24 hr following inoculation, a proinflammatory signature that lacked antiviral restriction factors was observed in viral RNA-positive tissues. The early innate response included expression of NLRX1, which inhibits antiviral responses, and activation of the TGF-ß pathway, which negatively regulates adaptive immune responses. These data suggest a model in which the virus triggers specific host mechanisms that suppress the generation of antiviral innate and adaptive immune responses in the first few days of infection, thus facilitating its own replication. These findings have important implications for the development of vaccines and other strategies to prevent infection.

Ad26/MVA therapeutic vaccination with TLR7 stimulation in SIV-infected rhesus monkeys.

The development of immunologic interventions that can target the viral reservoir in HIV-1-infected individuals is a major goal of HIV-1 research. However, little evidence exists that the viral reservoir can be sufficiently targeted to improve virologic control following discontinuation of antiretroviral therapy. Here we show that therapeutic vaccination with Ad26/MVA (recombinant adenovirus serotype 26 (Ad26) prime, modified vaccinia Ankara (MVA) boost) and stimulation of TLR7 (Toll-like receptor 7) improves virologic control and delays viral rebound following discontinuation of antiretroviral therapy in SIV-infected rhesus monkeys that began antiretroviral therapy during acute infection. Therapeutic vaccination with Ad26/MVA resulted in a marked increase in the magnitude and breadth of SIV-specific cellular immune responses in virologically suppressed, SIV-infected monkeys. TLR7 agonist administration led to innate immune stimulation and cellular immune activation. The combination of Ad26/MVA vaccination and TLR7 stimulation resulted in decreased levels of viral DNA in lymph nodes and peripheral blood, and improved virologic control and delayed viral rebound following discontinuation of antiretroviral therapy. The breadth of cellular immune responses correlated inversely with set point viral loads and correlated directly with time to viral rebound. These data demonstrate the potential of therapeutic vaccination combined with innate immune stimulation as a strategy aimed at a functional cure for HIV-1 infection.

Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys.

The viral reservoir represents a critical challenge for human immunodeficiency virus type 1 (HIV-1) eradication strategies. However, it remains unclear when and where the viral reservoir is seeded during acute infection and the extent to which it is susceptible to early antiretroviral therapy (ART). Here we show that the viral reservoir is seeded rapidly after mucosal simian immunodeficiency virus (SIV) infection of rhesus monkeys and before systemic viraemia. We initiated suppressive ART in groups of monkeys on days 3, 7, 10 and 14 after intrarectal SIVMAC251 infection. Treatment with ART on day 3 blocked the emergence of viral RNA and proviral DNA in peripheral blood and also substantially reduced levels of proviral DNA in lymph nodes and gastrointestinal mucosa as compared with treatment at later time points. In addition, treatment on day 3 abrogated the induction of SIV-specific humoral and cellular immune responses. Nevertheless, after discontinuation of ART following 24 weeks of fully suppressive therapy, virus rebounded in all animals, although the monkeys that were treated on day 3 exhibited a delayed viral rebound as compared with those treated on days 7, 10 and 14. The time to viral rebound correlated with total viraemia during acute infection and with proviral DNA at the time of ART discontinuation. These data demonstrate that the viral reservoir is seeded rapidly after intrarectal SIV infection of rhesus monkeys, during the 'eclipse' phase, and before detectable viraemia. This strikingly early seeding of the refractory viral reservoir raises important new challenges for HIV-1 eradication strategies.

Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys.

Human immunodeficiency virus type 1 (HIV-1)-specific monoclonal antibodies with extraordinary potency and breadth have recently been described. In humanized mice, combinations of monoclonal antibodies have been shown to suppress viraemia, but the therapeutic potential of these monoclonal antibodies has not yet been evaluated in primates with an intact immune system. Here we show that administration of a cocktail of HIV-1-specific monoclonal antibodies, as well as the single glycan-dependent monoclonal antibody PGT121, resulted in a rapid and precipitous decline of plasma viraemia to undetectable levels in rhesus monkeys chronically infected with the pathogenic simian-human immunodeficiency virus SHIV-SF162P3. A single monoclonal antibody infusion afforded up to a 3.1 log decline of plasma viral RNA in 7 days and also reduced proviral DNA in peripheral blood, gastrointestinal mucosa and lymph nodes without the development of viral resistance. Moreover, after monoclonal antibody administration, host Gag-specific T-lymphocyte responses showed improved functionality. Virus rebounded in most animals after a median of 56 days when serum monoclonal antibody titres had declined to undetectable levels, although, notably, a subset of animals maintained long-term virological control in the absence of further monoclonal antibody infusions. These data demonstrate a profound therapeutic effect of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys as well as an impact on host immune responses. Our findings strongly encourage the investigation of monoclonal antibody therapy for HIV-1 in humans.

Adenovirus serotype 26 and 35 vectors induce simian immunodeficiency virus-specific T lymphocyte responses in foreskin in rhesus monkeys.

UNLABELLED: Foreskin is the principal site of heterosexual HIV-1 infection in men. However, little is known about HIV-1-specific immune responses or inflammation in foreskin. To the best of our knowledge, no previous studies have assessed immune responses to candidate HIV-1 vaccines in foreskin. Using the rhesus monkey model, we show that intramuscular immunization with adenovirus serotype 26 and 35 vectors expressing SIV antigens elicited durable SIV Gag-specific CD4(+) and CD8(+) T cell responses in foreskin that were detectable for more than 1 year following vaccination. Gag-specific CD4(+) and CD8(+) T cells were also detectable in foreskin of SIV- and SHIV-infected animals and were at least comparable in magnitude to those in peripheral blood. However, unlike peripheral blood T cells, the majority of foreskin T cells exhibited transitional memory or effector memory phenotype and expressed higher levels of the activation markers CD69, HLA-DR, and CCR5, although vaccination did not further enhance foreskin CD4(+) T cell activation. These findings suggest that systemic vaccination strategies can elicit potentially important SIV-specific cellular immunity in foreskin. Further characterization of vaccine-elicited immune responses and inflammation in foreskin is warranted. IMPORTANCE: We demonstrate here the induction of SIV-specific cellular immune responses in foreskin by adenovirus serotype 26 and 35 vaccine vectors. Foreskin T cells were more activated than peripheral blood T cells, but foreskin T cells were not further activated by vaccination. These findings suggest that alternative serotype adenovirus vectors induce potentially important immune responses in foreskin.

Development of novel replication-defective lymphocytic choriomeningitis virus vectors expressing SIV antigens.

An important focus in vaccine research is the design of vaccine vectors with low seroprevalence and high immunogenicity. Replication-incompetent lymphocytic choriomeningitis virus (rLCMV) vectors do not elicit vector-neutralizing antibody responses, and homologous prime-boost regimens with rLCMV vectors induce boostable and protective T cell responses to model antigens in mice. However, cellular and humoral immune responses following homologous rLCMV vaccine regimens have not been rigorously evaluated in non-human primates (NHPs). To test whether rLCMV vectors constitute an effective vaccine platform in NHPs, we developed rLCMV vectors expressing SIVmac239 Env and Gag antigens and assessed their immunogenicity in mice and cynomolgus macaques. Immunization with rLCMV vaccine vectors expressing SIV Env and Gag was effective at generating SIV-specific T cell and antibody responses in both mice and NHPs. Epitope mapping using SIV Env in C57BL/6 mice demonstrated that rLCMV vectors induced sustained poly-functional responses to both dominant and subdominant epitopes. Our results suggest the potential of rLCMV vectors as vaccine candidates. Future SIV challenge experiments in rhesus macaques will be needed to assess immune protection by these vaccine vectors.

Antibody-mediated protection against SHIV challenge includes systemic clearance of distal virus.

HIV-1-specific broadly neutralizing antibodies (bNAbs) can protect rhesus monkeys against simian-human immunodeficiency virus (SHIV) challenge. However, the site of antibody interception of virus and the mechanism of antibody-mediated protection remain unclear. We administered a fully protective dose of the bNAb PGT121 to rhesus monkeys and challenged them intravaginally with SHIV-SF162P3. In PGT121-treated animals, we detected low levels of viral RNA and viral DNA in distal tissues for seven days following challenge. Viral RNA-positive tissues showed transcriptomic changes indicative of innate immune activation, and cells from these tissues initiated infection after adoptive transfer into naïve hosts. These data demonstrate that bNAb-mediated protection against a mucosal virus challenge can involve clearance of infectious virus in distal tissues.

Protective efficacy of multiple vaccine platforms against Zika virus challenge in rhesus monkeys.

Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causally associated with fetal microcephaly. The development of a safe and effective ZIKV vaccine is therefore an urgent global health priority. Here we demonstrate that three different vaccine platforms protect against ZIKV challenge in rhesus monkeys. A purified inactivated virus vaccine induced ZIKV-specific neutralizing antibodies and completely protected monkeys against ZIKV strains from both Brazil and Puerto Rico. Purified immunoglobulin from vaccinated monkeys also conferred passive protection in adoptive transfer studies. A plasmid DNA vaccine and a single-shot recombinant rhesus adenovirus serotype 52 vector vaccine, both expressing ZIKV premembrane and envelope, also elicited neutralizing antibodies and completely protected monkeys against ZIKV challenge. These data support the rapid clinical development of ZIKV vaccines for humans.