Establishment and recall of SARS-CoV-2 spike epitope-specific CD4+ T cell memory.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination elicit CD4+ T cell responses to the spike protein, including circulating follicular helper T (cTFH) cells that correlate with neutralizing antibodies. Using a novel HLA-DRB1*15:01/S751 tetramer to track spike-specific CD4+ T cells, we show that primary infection or vaccination induces robust S751-specific CXCR5- and cTFH cell memory responses. Secondary exposure induced recall of CD4+ T cells with a transitory CXCR3+ phenotype, and drove expansion of cTFH cells transiently expressing ICOS, CD38 and PD-1. In both contexts, cells exhibited a restricted T cell antigen receptor repertoire, including a highly public clonotype and considerable clonotypic overlap between CXCR5- and cTFH populations. Following a third vaccine dose, the rapid re-expansion of spike-specific CD4+ T cells contrasted with the comparatively delayed increase in antibody titers. Overall, we demonstrate that stable pools of cTFH and memory CD4+ T cells established by infection and/or vaccination are efficiently recalled upon antigen reexposure and may contribute to long-term protection against SARS-CoV-2.

SARS-CoV-2 breakthrough infection induces rapid memory and de novo T cell responses.

Although the protective role of neutralizing antibodies against COVID-19 is well established, questions remain about the relative importance of cellular immunity. Using 6 pMHC multimers in a cohort with early and frequent sampling, we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection in previously vaccinated individuals. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable degrees of expansion. The frequency of activated SARS-CoV-2-specific CD8+ T cells at baseline and peak inversely correlated with peak SARS-CoV-2 RNA levels in nasal swabs and accelerated viral clearance. Our study demonstrates that a rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

MAIT Cells Upregulate α4ß7 in Response to Acute Simian Immunodeficiency Virus/Simian HIV Infection but Are Resistant to Peripheral Depletion in Pigtail Macaques.

Mucosal-associated invariant T (MAIT) cells are nonconventional T lymphocytes that recognize bacterial metabolites presented by MR1. Whereas gut bacterial translocation and the loss/dysfunction of peripheral MAIT cells in HIV infection is well described, MAIT cells in nonhuman primate models are poorly characterized. We generated a pigtail macaque (PTM)-specific MR1 tetramer and characterized MAIT cells in serial samples from naive and SIV- or simian HIV-infected PTM. Although PTM MAIT cells generally resemble the phenotype and transcriptional profile of human MAIT cells, they exhibited uniquely low expression of the gut-homing marker α4ß7 and were not enriched at the gut mucosa. PTM MAIT cells responded to SIV/simian HIV infection by proliferating and upregulating α4ß7, coinciding with increased MAIT cell frequency in the rectum. By 36 wk of infection, PTM MAIT cells were activated and exhibited a loss of Tbet expression but were not depleted as in HIV infection. Our data suggest the following: 1) MAIT cell activation and exhaustion is uncoupled from the hallmark depletion of MAIT cells during HIV infection; and 2) the lack of PTM MAIT cell enrichment at the gut mucosa may prevent depletion during chronic infection, providing a model to assess potential immunotherapeutic approaches to modify MAIT cell trafficking during HIV infection.

MAIT cells are depleted early but retain functional cytokine expression in HIV infection.

Mucosal-associated invariant T (MAIT) cells home to mucosal sites and exert antimicrobial activity against bacteria and other microorganisms. HIV infection leads to early depletion of gut T cells and translocation of bacterial products. There are reports that MAIT cells, defined by coexpression of Vα7.2 and CD161, are depleted during HIV infection and residual MAIT cells are functionally impaired. However, one study suggested that MAIT cells might remain after HIV infection but evade detection through CD161 downregulation. Thus, the impact of HIV infection on MAIT cells is unclear. We studied longitudinal blood samples from 31 HIV-infected subjects for MAIT cell numbers, phenotype and function using both standard Vα7.2/CD161 surface markers and an MR1 tetramer. We found that MAIT cells were depleted early during HIV infection, and although there was a concomitant rise in Vα7.2(+)CD161(-) cells, these were MR1 tetramer negative, indicating that these are unlikely to be altered MAIT cells. Antigen-mediated activation of residual MAIT cells showed that they remained functional out to 2 years following HIV infection. Although MAIT cells are depleted in HIV infection, residual and functionally active MAIT cells persist and may still be able to assist in controlling bacterial translocation during HIV infection.

Standard trivalent influenza virus protein vaccination does not prime antibody-dependent cellular cytotoxicity in macaques.

Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended, since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce antibody-dependent cellular cytotoxicity (ADCC) responses that can cross-recognize divergent influenza virus strains is unknown. We immunized 6 influenza-naive pigtail macaques twice with the 2011-2012 season TIV and then challenged the macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza virus-specific CD8 T cell (CTL) responses using a sensitive major histocompatibility complex (MHC) tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA), did not induce detectable influenza virus-specific ADCC or CTL responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins, but not influenza virus HA proteins from different subtypes (H2 to H7). There was no anamnestic influenza virus-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or to divergent H3 proteins but did boost CTL responses. ADCC or CTL responses were not induced by TIV vaccination in influenza-naive macaques. There was a marked difference in the ability of infection compared to that of vaccination to induce cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.

Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1.

BACKGROUND: During the 2009 pandemic of influenza A virus subtype H1N1 (A[H1N1]pdm09) infection, older individuals were partially protected from severe disease. It is not known whether preexisting antibodies with effector functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed. METHODS: We tested serum specimens obtained from 182 individuals aged 1-72 years that were collected either immediately before or after the A(H1N1)pdm09 pandemic for ADCC antibodies to the A(H1N1)pdm09 hemagglutinin (HA) protein. RESULTS: A(H1N1)pdm09 HA-specific ADCC antibodies were detected in almost all individuals aged >45 years (28/31 subjects) before the 2009 A(H1N1) pandemic. Conversely, only approximately half of the individuals aged 1-14 years (11/31) and 15-45 years (17/31) had cross-reactive ADCC antibodies before the 2009 A(H1N1) pandemic. The A(H1N1)pdm09-specific ADCC antibodies were able to efficiently mediate the killing of influenza virus-infected respiratory epithelial cells. Further, subjects >45 years of age had higher ADCC titers to a range of seasonal H1N1 HA proteins, including from the 1918 virus, compared with younger individuals. CONCLUSIONS: ADCC antibodies may have contributed to the protection exhibited in older individuals during the 2009 A(H1N1) pandemic. This work has significant implications for improved vaccination strategies for future influenza pandemics.

Phenotypic and functional characterization of pharmacologically expanded Vγ9Vδ2 T cells in pigtail macaques.

While gaining interest as treatment for cancer and infectious disease, the clinical efficacy of Vγ9Vδ2 T cell-based immunotherapeutics has to date been limited. An improved understanding of γδ T cell heterogeneity across lymphoid and non-lymphoid tissues, before and after pharmacological expansion, is required. Here, we describe the phenotype and tissue distribution of Vγ9Vδ2 T cells at steady state and following in vivo pharmacological expansion in pigtail macaques. Intravenous phosphoantigen administration with subcutaneous rhIL-2 drove robust expansion of Vγ9Vδ2 T cells in blood and pulmonary mucosa, while expansion was confined to the pulmonary mucosa following intratracheal antigen administration. Peripheral blood Vγ9Vδ2 T cell expansion was polyclonal, and associated with a significant loss of CCR6 expression due to IL-2-mediated receptor downregulation. Overall, we show the tissue distribution and phenotype of in vivo pharmacologically expanded Vγ9Vδ2 T cells can be altered based on the antigen administration route, with implications for tissue trafficking and the clinical efficacy of Vγ9Vδ2 T cell immunotherapeutics.

Durable reprogramming of neutralizing antibody responses following Omicron breakthrough infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection of vaccinated individuals is increasingly common with the circulation of highly immune evasive and transmissible Omicron variants. Here, we report the dynamics and durability of recalled spike-specific humoral immunity following Omicron BA.1 or BA.2 breakthrough infection, with longitudinal sampling up to 8 months after infection. Both BA.1 and BA.2 infections robustly boosted neutralization activity against the infecting strain while expanding breadth against BA.4, although neutralization activity was substantially reduced for the more recent XBB and BQ.1.1 strains. Cross-reactive memory B cells against both ancestral and Omicron spike were predominantly expanded by infection, with limited recruitment of de novo Omicron-specific B cells or antibodies. Modeling of neutralization titers predicts that protection from symptomatic reinfection against antigenically similar strains will be durable but is undermined by new emerging strains with further neutralization escape.

Anti-Drug Antibodies in Pigtailed Macaques Receiving HIV Broadly Neutralising Antibody PGT121.

Broadly neutralising antibodies (bNAbs) may play an important role in future strategies for HIV control. The development of anti-drug antibody (ADA) responses can reduce the efficacy of passively transferred bNAbs but the impact of ADA is imperfectly understood. We previously showed that therapeutic administration of the anti-HIV bNAb PGT121 (either WT or LALA version) controlled viraemia in pigtailed macaques with ongoing SHIV infection. We now report on 23 macaques that had multiple treatments with PGT121. We found that an increasing number of intravenous doses of PGT121 or human IgG1 isotype control antibodies (2-4 doses) results in anti-PGT121 ADA induction and low plasma concentrations of PGT121. ADA was associated with poor or absent suppression of SHIV viremia. Notably, ADA within macaque plasma recognised another human bNAb 10E8 but did not bind to the variable domains of PGT121, suggesting that ADA were primarily directed against the constant regions of the human antibodies. These findings have implications for the development of preclinical studies examining multiple infusions of human bNAbs.

Interactions of core cross-linked poly(2-oxazoline) and poly(2-oxazine) micelles with immune cells in human blood.

Water-soluble poly(cyclic imino ether)s (PCIEs) have emerged as promising biocompatible polymers for nanomedicine applications in recent years. Despite their generally accepted stealth properties, there has been no comprehensive evaluation of their interactions with primary immune cells in human blood. Here we present a library of core cross-linked micelles (CCMs) containing various PCIE shells. Well-defined high molar mass CCMs (Mn > 175 kDa, Р< 1.2) of similar diameter (~20 nm) were synthesised using a cationic ring-opening polymerisation (CROP) - surfactant-free reversible addition-fragmentation chain-transfer (RAFT) emulsion polymerisation strategy. The stealth properties of the different PCIE CCMs were assessed employing a whole human blood assay simulating the complex blood environment. Cell association studies revealed lower associations of poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) CCMs with blood immune cells compared to the respective poly(2-oxazine) (POz) CCMs. Noteworthy, PMeOx CCMs outperformed all other reported CCMs, showing overall low associations and only negligible differences in the presence and absence of serum proteins. This study highlights the importance of investigating individual nanomaterials under physiologically relevant conditions and further strengthens the position of PMeOx as a highly promising stealth material for biomedical applications.

Influence of Poly(ethylene glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle-Immune Cell Interactions in Human Blood.

Poly(ethylene glycol) (PEG) is widely used in particle assembly to impart biocompatibility and stealth-like properties in vivo for diverse biomedical applications. Previous studies have examined the effect of PEG molecular weight and PEG coating density on the biological fate of various particles; however, there are few studies that detail the fundamental role of PEG molecular architecture in particle engineering and bio-nano interactions. Herein, we engineered PEG particles using a mesoporous silica (MS) templating method and investigated how the PEG building block architecture impacted the physicochemical properties (e.g., surface chemistry and mechanical characteristics) of the PEG particles and subsequently modulated particle-immune cell interactions in human blood. Varying the PEG architecture from 3-arm to 4-arm, 6-arm, and 8-arm generated PEG particles with a denser, stiffer structure, with increasing elastic modulus from 1.5 to 14.9 kPa, inducing an increasing level of immune cell association (from 15% for 3-arm to 45% for 8-arm) with monocytes. In contrast, the precursor PEG particles with the template intact (MS@PEG) were stiffer and generally displayed higher levels of immune cell association but showed the opposite trend-immune cell association decreased with increasing PEG arm numbers. Proteomics analysis demonstrated that the biomolecular corona that formed on the PEG particles minimally influenced particle-immune cell interactions, whereas the MS@PEG particle-cell interactions correlated with the composition of the corona that was abundant in histidine-rich glycoproteins. Our work highlights the role of PEG architecture in the design of stealth PEG-based particles, thus providing a link between the synthetic nature of particles and their biological behavior in blood.

Immunogenicity of prime-boost protein subunit vaccine strategies against SARS-CoV-2 in mice and macaques.

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assess prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD is associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augments neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines are comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.

Protective efficacy of the anti-HIV broadly neutralizing antibody PGT121 in the context of semen exposure.

BACKGROUND: HIV-1 infections occur following viral exposure at anogenital mucosal surfaces in the presence of semen. Semen contains immunosuppressive and pro-inflammatory factors. Semen from HIV-1-infected donors contains anti-HIV-1 antibodies. We assessed if passively infused anti-HIV-1 neutralizing antibody conferred protection from rectal SHIVSF162P3 challenge at semen exposed mucosae. METHODS: We pooled seminal plasma from HIV-1-infected donors. The pool was screened by ELISA for antibodies against HIV-1SF162 gp140. The ability of seminal plasma to inhibit macaque NK cells from responding to direct and antibody-dependent stimulation was assessed. The ability of seminal plasma to inhibit macaque granulocytes from mediating oxidative burst was also assessed. To demonstrate viral infectivity in the presence of seminal plasma, macaques (n = 4) were rectally challenged with SHIVSF162P3 following exposure to 2.5 mL of seminal plasma. To evaluate if anti-HIV-1 neutralizing antibody confers protection against rectal SHIV challenge at semen exposed mucosae, eight macaques were intravenously infused with PGT121, either wild type (n = 4) or the Fc receptor binding deficient LALA variant (n = 4), and rectally challenged with SHIVSF162P3 following exposure to 2.5 mL of seminal plasma. FINDINGS: Anti-HIV-1SF162 gp140 antibodies were detected in seminal plasma. Seminal plasma inhibited direct and antibody-dependent NK cell activation and granulocyte oxidative burst in vitro. Rectal SHIVSF162P3 challenge of control macaques following seminal plasma exposure resulted in infection of all animals. All macaques infused with wild type or LALA PGT121 and challenged with SHIVSF162P3 following seminal plasma exposure were protected. INTERPRETATION: PGT121 conferred protection against rectal SHIVSF162P3 challenge at semen exposed mucosae. Future research should investigate if semen alters protection conferred by antibodies more dependent on non-neutralizing functions. FUNDING: This work was supported by a grant from the Australian National Health and Medical Research Council (APP1124680).

Adaptive immunity to human coronaviruses is widespread but low in magnitude.

OBJECTIVES: Endemic human coronaviruses (hCoVs) circulate worldwide but cause minimal mortality. Although seroconversion to hCoV is near ubiquitous during childhood, little is known about hCoV-specific T-cell memory in adults. METHODS: We quantified CD4 T-cell and antibody responses to hCoV spike antigens in 42 SARS-CoV-2-uninfected individuals. Antigen-specific memory T cells and circulating T follicular helper (cTFH) cells were identified using an activation-induced marker assay and characterised for memory phenotype and chemokine receptor expression. RESULTS: T-cell responses were widespread within conventional memory and cTFH compartments but did not correlate with IgG titres. SARS-CoV-2 cross-reactive T cells were observed in 48% of participants and correlated with HKU1 memory. hCoV-specific T cells exhibited a CCR6+ central memory phenotype in the blood, but were enriched for frequency and CXCR3 expression in human lung-draining lymph nodes. CONCLUSION: Overall, hCoV-specific humoral and cellular memory are independently maintained, with a shared phenotype existing among coronavirus-specific CD4 T cells. This understanding of endemic coronavirus immunity provides insight into the homeostatic maintenance of immune responses that are likely to be critical components of protection against SARS-CoV-2.

Landscape of human antibody recognition of the SARS-CoV-2 receptor binding domain.

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.

Mucosal and systemic SIV-specific cytotoxic CD4+ T cell hierarchy in protection following intranasal/intramuscular recombinant pox-viral vaccination of pigtail macaques.

A HIV vaccine that provides mucosal immunity is urgently needed. We evaluated an intranasal recombinant Fowlpox virus (rFPV) priming vaccine followed by intramuscular Modified Vaccinia Ankara (rMVA) booster vaccine, both expressing SIV antigens. The vaccination generated mucosal and systemic SIV-specific CD4+ T cell mediated immunity and was associated with partial protection against high-dose intrarectal SIVmac251 challenge in outbred pigtail macaques. Three of 12 vaccinees were completely protected and these animals elicited sustained Gag-specific poly-functional, cytotoxic mucosal CD4+ T cells, complemented by systemic poly-functional CD4+ and CD8+ T cell immunity. Humoral immune responses, albeit absent in completely protected macaques, were associated with partial control of viremia in animals with relatively weaker mucosal/systemic T cell responses. Co-expression of an IL-4R antagonist by the rFPV vaccine further enhanced the breadth and cytotoxicity/poly-functionality of mucosal vaccine-specific CD4+ T cells. Moreover, a single FPV-gag/pol/env prime was able to induce rapid anamnestic gp140 antibody response upon SIV encounter. Collectively, our data indicated that nasal vaccination was effective at inducing robust cervico-vaginal and rectal immunity, although cytotoxic CD4+ T cell mediated mucosal and systemic immunity correlated strongly with 'complete protection', the different degrees of protection observed was multi-factorial.

Fc-dependent functions are redundant to efficacy of anti-HIV antibody PGT121 in macaques.

A considerable body of evidence suggests that Fc-dependent functions improve the capacity of broadly neutralizing antibodies (BnAbs) to protect against and control HIV-1 infection. This phenomenon, however, has not been formally tested in robust cell-associated macaque simian-human immunodeficiency virus (SHIV) models with newer-generation BnAbs. We studied both the WT BnAb PGT121 and a LALA mutant of PGT121 (which has impaired Fc-dependent functions) for their ability to protect pigtail macaques from an i.v. high-dose cell-associated SHIVSF162P3 challenge. We found that both WT and LALA PGT121 completely protected all 12 macaques studied. Further, partial depletion of NK cells, key mediators of Fc-dependent functions, did not abrogate the protective efficacy of PGT121 in 6 macaques. Additionally, in animals with established SHIVSF162P3 infection, SHIV viremia levels were equally rapidly reduced by LALA and WT PGT121. Our studies suggest that the potent neutralizing capacity of PGT121 renders the Fc-dependent functions of the Ab at least partially redundant. These findings have implications for Ab-mediated protection from and control of HIV-1 infection.

Partial efficacy of a broadly neutralizing antibody against cell-associated SHIV infection.

Broadly neutralizing antibodies (BnAbs) protect macaques from cell-free simian/human immunodeficiency virus (SHIV) challenge, but their efficacy against cell-associated SHIV is unclear. Virus in cell-associated format is highly infectious, present in transmission-competent bodily fluids, and potentially capable of evading antibody-mediated neutralization. The PGT121 BnAb, which recognizes an epitope consisting of the V3 loop and envelope glycans, mediates antibody-dependent cellular cytotoxicity and neutralization of cell-to-cell HIV-1 transmission. To evaluate whether a BnAb can prevent infection after cell-associated viral challenge, we infused pigtail macaques with PGT121 or an isotype control and challenged animals 1 hour later intravenously with SHIVSF162P3-infected splenocytes. All five controls had high viremia 1 week after challenge. Three of six PGT121-infused animals were completely protected, two of six animals had a 1-week delay in onset of high viremia, and one animal had a 7-week delay in onset of viremia. The infused antibody had decayed on average to 2.0 µg/ml by 1 week after infusion and was well below 1 µg/ml (range, <0.1 to 0.8 µg/ml) by 8 weeks. The animals with a 1-week delay before high viremia had relatively lower plasma concentrations of PGT121. Transfer of 22 million peripheral blood mononuclear cells (PBMCs) stored at weeks 1 to 4 from the animal with the 7-week delayed onset of viremia into uninfected macaques did not initiate infection. Our results show that HIV-1-specific neutralizing antibodies have partial efficacy against cell-associated virus exposure in macaques. We conclude that sustaining high concentrations of bioavailable BnAb is important for protecting against cell-associated virus.

Recombinant influenza virus expressing HIV-1 p24 capsid protein induces mucosal HIV-specific CD8 T-cell responses.

Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection.

High fidelity simian immunodeficiency virus reverse transcriptase mutants have impaired replication in vitro and in vivo.

The low fidelity of HIV replication facilitates immune and drug escape. Some reverse transcriptase (RT) inhibitor drug-resistance mutations increase RT fidelity in biochemical assays but their effect during viral replication is unclear. We investigated the effect of RT mutations K65R, Q151N and V148I on SIV replication and fidelity in vitro, along with SIV replication in pigtailed macaques. SIVmac239-K65R and SIVmac239-V148I viruses had reduced replication capacity compared to wild-type SIVmac239. Direct virus competition assays demonstrated a rank order of wild-type>K65R>V148I mutants in terms of viral fitness. In single round in vitro-replication assays, SIVmac239-K65R demonstrated significantly higher fidelity than wild-type, and rapidly reverted to wild-type following infection of macaques. In contrast, SIVmac239-Q151N was replication incompetent in vitro and in pigtailed macaques. Thus, we showed that RT mutants, and specifically the common K65R drug-resistance mutation, had impaired replication capacity and higher fidelity. These results have implications for the pathogenesis of drug-resistant HIV.