The Frequency of Vaccine-Induced T-Cell Responses Does Not Predict the Rate of Acquisition after Repeated Intrarectal SIVmac239 Challenges in Mamu-B*08+ Rhesus Macaques.

Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic-phase viremia after infection with the pathogenic simian immunodeficiency virus mac239 (SIVmac239) clone. CD8+ T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08-positive (Mamu-B*08+ ) RMs. Here we evaluated if robust vaccine-elicited CD8+ T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08+ RMs following mucosal SIV challenges. Ten Mamu-B*08+ RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8+ T cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsy specimens. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges, and three of them remained aviremic after 3 or 4 additional challenges. The rate of SIV acquisition in the vaccinees was numerically lower (albeit not statistically significantly) than that in the controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection from those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection.IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8+ T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a lower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8+ T cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection.

Mamu-B*17+ Rhesus Macaques Vaccinated with env, vif, and nef Manifest Early Control of SIVmac239 Replication.

Certain major histocompatibility complex class I (MHC-I) alleles are associated with spontaneous control of viral replication in human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs). These cases of "elite" control of HIV/SIV replication are often immune-mediated, thereby providing a framework for studying anti-lentiviral immunity. In this study, we examined how vaccination impacts SIV replication in RMs expressing the MHC-I allele Mamu-B*17 Approximately 21% of Mamu-B*17+ and 50% of Mamu-B*08+ RMs control chronic-phase viremia after SIVmac239 infection. Because CD8+ T cells targeting Mamu-B*08-restricted SIV epitopes have been implicated in virologic suppression in Mamu-B*08+ RMs, we investigated whether this might also be true for Mamu-B*17+ RMs. Two groups of Mamu-B*17+ RMs were vaccinated with genes encoding Mamu-B*17-restricted epitopes in Vif and Nef. These genes were delivered by themselves (group 1) or together with env (group 2). Group 3 included MHC-I-matched RMs and served as the control group. Surprisingly, the group 1 vaccine regimen had little effect on viral replication compared to group 3, suggesting that unlike Mamu-B*08+ RMs, preexisting SIV-specific CD8+ T cells alone do not facilitate long-term virologic suppression in Mamu-B*17+ RMs. Remarkably, however, 5/8 group 2 vaccinees controlled viremia to <15 viral RNA copies/ml soon after infection. No serological neutralizing activity against SIVmac239 was detected in group 2, although vaccine-elicited gp140-binding antibodies correlated inversely with nadir viral loads. Collectively, these data shed new light on the unique mechanism of elite control in Mamu-B*17+ RMs and implicate vaccine-induced, nonneutralizing anti-Env antibodies in the containment of immunodeficiency virus infection.IMPORTANCE A better understanding of the immune correlates of protection against HIV might facilitate the development of a prophylactic vaccine. Therefore, we investigated simian immunodeficiency virus (SIV) infection outcomes in rhesus macaques expressing the major histocompatibility complex class I allele Mamu-B*17 Approximately 21% of Mamu-B*17+ macaques spontaneously controlled chronic phase viremia after SIV infection, an effect that may involve CD8+ T cells targeting Mamu-B*17-restricted SIV epitopes. We vaccinated Mamu-B*17+ macaques with genes encoding immunodominant epitopes in Vif and Nef alone (group 1) or together with env (group 2). Although neither vaccine regimen prevented SIV infection, 5/8 group 2 vaccinees controlled viremia to below detection limits shortly after infection. This outcome, which was not observed in group 1, was associated with vaccine-induced, nonneutralizing Env-binding antibodies. Together, these findings suggest a limited contribution of Vif- and Nef-specific CD8+ T cells for virologic control in Mamu-B*17+ macaques and implicate anti-Env antibodies in containment of SIV infection.

Vaccine-induced immune responses against both Gag and Env improve control of simian immunodeficiency virus replication in rectally challenged rhesus macaques.

The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.

Potent Plasmablast-Derived Antibodies Elicited by the National Institutes of Health Dengue Vaccine.

Exposure to dengue virus (DENV) is thought to elicit lifelong immunity, mediated by DENV-neutralizing antibodies (nAbs). However, Abs generated by primary infections confer serotype-specific protection, and immunity against other serotypes develops only after subsequent infections. Accordingly, the induction of these nAb responses acquired after serial DENV infections has been a long-sought-after goal for vaccination. Nonetheless, it is still unclear if tetravalent vaccines can elicit or recall nAbs. In this study, we have characterized the responses from a volunteer who had been previously exposed to DENV and was immunized with the live attenuated tetravalent vaccine Butantan-DV, developed by the NIH and Butantan Institute. Eleven days after vaccination, we observed an ∼70-fold expansion of the plasmablast population. We generated 21 monoclonal Abs (MAbs) from singly sorted plasmablasts. These MAbs were the result of clonal expansions and had significant levels of somatic hypermutation (SHM). Nineteen MAbs (90.5%) neutralized at least one DENV serotype at concentrations of 1 µg/ml or less; 6 of the 21 MAbs neutralized three or more serotypes. Despite the tetravalent composition of the vaccine, we observed a neutralization bias in the induced repertoire: DENV3 was targeted by 18 of the 19 neutralizing MAbs (nMAbs). Furthermore, the P3D05 nMAb neutralized DENV3 with extraordinary potency (concentration to achieve half-maximal neutralization [Neut50] = 0.03 µg/ml). Thus, the Butantan-DV vaccine engendered a mature, antigen-selected B cell repertoire. Our results suggest that preexisting responses elicited by a previous DENV3 infection were recalled by immunization.IMPORTANCE The dengue epidemic presents a global public health challenge that causes widespread economic burden and remains largely unchecked by existing control strategies. Successful control of the dengue epidemic will require effective prophylactic and therapeutic interventions. Several vaccine clinical efficacy trials are approaching completion, and the chances that one or more live attenuated tetravalent vaccines (LATVs) will be introduced worldwide is higher than ever. While it is widely accepted that dengue virus (DENV)-neutralizing antibody (nAb) titers are associated with protection, the Ab repertoire induced by LATVs remain uncharacterized. Here, we describe the isolation of potent (Neut50 < 0.1 µg/ml) nAbs from a DENV-seropositive volunteer immunized with the tetravalent vaccine Butantan-DV, which is currently in phase III trials.

Dengue Virus Evades AAV-Mediated Neutralizing Antibody Prophylaxis in Rhesus Monkeys.

Development of vaccines against mosquito-borne Flaviviruses is complicated by the occurrence of antibody-dependent enhancement (ADE), which can increase disease severity. Long-term delivery of neutralizing antibodies (nAbs) has the potential to effectively block infection and represents an alternative to vaccination. The risk of ADE may be avoided by using prophylactic nAbs harboring amino acid mutations L234A and L235A (LALA) in the immunoglobulin G (IgG) constant region. Here, we used recombinant adeno-associated viruses (rAAVs) to deliver the anti-dengue virus 3 (DENV3) nAb P3D05. While the administration of rAAV-P3D05-rhesus immunoglobulin G1 (rhIgG1)-LALA to rhesus macaques engendered DENV3-neutralizing activity in serum, it did not prevent infection. The emergence of viremia following DENV3 challenge was delayed by 3-6 days in the rAAV-treated group, and replicating virus contained the envelope mutation K64R. This neutralization-resistant variant was also confirmed by virus outgrowth experiments in vitro. By delivering P3D05 with unmutated Fc sequences, we further demonstrated that DENV3 also evaded wild-type nAb prophylaxis, and serum viral loads appeared to be higher in the presence of low levels of unmutated P3D05-rhIgG1. Our study shows that a vectored approach for long-term delivery of nAbs with the LALA mutations is promising, but prophylaxis using a single nAb is likely insufficient at preventing DENV infection and replication.

Induction of Transient Virus Replication Facilitates Antigen-Independent Isolation of SIV-Specific Monoclonal Antibodies.

Structural characterization of the HIV-1 Envelope (Env) glycoprotein has facilitated the development of Env probes to isolate HIV-specific monoclonal antibodies (mAbs). However, preclinical studies have largely evaluated these virus-specific mAbs against chimeric viruses, which do not naturally infect non-human primates, in contrast to the unconstrained simian immunodeficiency virus (SIV)mac239 clone. Given the paucity of native-like reagents for the isolation of SIV-specific B cells, we examined a method to isolate SIVmac239-specific mAbs without using Env probes. We first activated virus-specific B cells by inducing viral replication after the infusion of a CD8ß-depleting mAb or withdrawal of antiretroviral therapy in SIVmac239-infected rhesus macaques. Following the rise in viremia, we observed 2- to 4-fold increases in the number of SIVmac239 Env-reactive plasmablasts in circulation. We then sorted these activated B cells and obtained 206 paired Ab sequences. After expressing 122 mAbs, we identified 14 Env-specific mAbs. While these Env-specific mAbs bound to both the SIVmac239 SOSIP.664 trimer and to infected primary rhesus CD4+ T cells, five also neutralized SIVmac316. Unfortunately, none of these mAbs neutralized SIVmac239. Our data show that this method can be used to isolate virus-specific mAbs without antigenic probes by inducing bursts of contemporary replicating viruses in vivo.

Fetal demise and failed antibody therapy during Zika virus infection of pregnant macaques.

Zika virus (ZIKV) infection of pregnant women is associated with pathologic complications of fetal development. Here, we infect pregnant rhesus macaques (Macaca mulatta) with a minimally passaged ZIKV isolate from Rio de Janeiro, where a high rate of fetal development complications was observed. The infection of pregnant macaques with this virus results in maternal viremia, virus crossing into the amniotic fluid (AF), and in utero fetal deaths. We also treated three additional ZIKV-infected pregnant macaques with a cocktail of ZIKV-neutralizing human monoclonal antibodies (nmAbs) at peak viremia. While the nmAbs can be effective in clearing the virus from the maternal sera of treated monkeys, it is not sufficient to clear ZIKV from AF. Our report suggests that ZIKV from Brazil causes fetal demise in non-human primates (NHPs) without additional mutations or confounding co-factors. Treatment with a neutralizing anti-ZIKV nmAb cocktail is insufficient to fully stop vertical transmission.

Rare Control of SIVmac239 Infection in a Vaccinated Rhesus Macaque.

Effector memory T cell (TEM) responses display potent antiviral properties and have been linked to stringent control of simian immunodeficiency virus (SIV) replication. Since recurrent antigen stimulation drives the differentiation of CD8+ T cells toward the TEM phenotype, in this study we incorporated a persistent herpesviral vector into a heterologous prime/boost/boost vaccine approach to maximize the induction of TEM responses. This new regimen resulted in CD8+ TEM-biased responses in four rhesus macaques, three of which controlled viral replication to <1,000 viral RNA copies/ml of plasma for more than 6 months after infection with SIVmac239. Over the course of this study, we made a series of interesting observations in one of these successful controller animals. Indeed, in vivo elimination of CD8αß+ T cells using a new CD8ß-depleting antibody did not abrogate virologic control in this monkey. Only after its CD8α+ lymphocytes were depleted did SIV rebound, suggesting that CD8αα+ but not CD8αß+ cells were controlling viral replication. By 2 weeks postinfection (PI), the only SIV sequences that could be detected in this animal harbored a small in-frame deletion in nef affecting six amino acids. Deep sequencing of the SIVmac239 challenge stock revealed no evidence of this polymorphism. However, sequencing of the rebound virus following CD8α depletion at week 38.4 PI again revealed only the six-amino acid deletion in nef. While any role for immunological pressure on the selection of this deleted variant remains uncertain, our data provide anecdotal evidence that control of SIV replication can be maintained without an intact CD8αß+ T cell compartment.

Ontogeny of the B- and T-cell response in a primary Zika virus infection of a dengue-naïve individual during the 2016 outbreak in Miami, FL.

Zika virus (ZIKV) is a mosquito-borne flavivirus of significant public health concern. In the summer of 2016, ZIKV was first detected in the contiguous United States. Here we present one of the first cases of a locally acquired ZIKV infection in a dengue-naïve individual. We collected blood from a female with a maculopapular rash at day (D) 5 and D7 post onset of symptoms (POS) and we continued weekly blood draws out to D148 POS. To establish the ontogeny of the immune response against ZIKV, lymphocytes and plasma were analyzed in a longitudinal fashion. The plasmablast response peaked at D7 POS (19.6% of CD19+ B-cells) and was undetectable by D15 POS. ZIKV-specific IgM was present at D5 POS, peaked between D15 and D21 POS, and subsequently decreased. The ZIKV-specific IgG response, however, was not detected until D15 POS and continued to increase after that. Interestingly, even though the patient had never been infected with dengue virus (DENV), cross-reactive IgM and IgG binding against each of the four DENV serotypes could be detected. The highest plasma neutralization activity against ZIKV peaked between D15 and D21 POS, and even though DENV binding antibodies were present in the plasma of the patient, there was neither neutralization nor antibody dependent enhancement (ADE) of DENV. Interestingly, ADE against ZIKV arose at D48 POS and continued until the end of the study. CD4+ and CD8+ T-cells recognized ZIKV-NS2A and ZIKV-E, respectively. The tetramer positive CD8+ T-cell response peaked at D21 POS with elevated levels persisting for months. In summary, this is the first study to establish the timing of the ontogeny of the immune response against ZIKV.

A human inferred germline antibody binds to an immunodominant epitope and neutralizes Zika virus.

The isolation of neutralizing monoclonal antibodies (nmAbs) against the Zika virus (ZIKV) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. Here we describe the characterization of human mAbs from the plasmablasts of an acutely infected patient. One of the 18 mAbs had the unusual feature of binding to and neutralizing ZIKV despite not appearing to have been diversified by affinity maturation. This mAb neutralized ZIKV (Neut50 ~ 2 µg/ml) but did not react with any of the four dengue virus serotypes. Except for the expected junctional diversity created by the joining of the V-(D)-J genes, there was no deviation from immunoglobulin germline genes. This is a rare example of a human mAb with neutralizing activity in the absence of detectable somatic hypermutation. Importantly, binding of this mAb to ZIKV was specifically inhibited by human plasma from ZIKV-exposed individuals, suggesting that it may be of value in a diagnostic setting.

Neutralizing human monoclonal antibodies prevent Zika virus infection in macaques.

Therapies to prevent maternal Zika virus (ZIKV) infection and its subsequent fetal developmental complications are urgently required. We isolated three potent ZIKV-neutralizing monoclonal antibodies (nmAbs) from the plasmablasts of a ZIKV-infected patient-SMZAb1, SMZAb2, and SMZAb5-directed against two different domains of the virus. We engineered these nmAbs with Fc LALA mutations that abrogate Fcγ receptor binding, thus eliminating potential therapy-mediated antibody-dependent enhancement. We administered a cocktail of these three nmAbs to nonhuman primates 1 day before challenge with ZIKV and demonstrated that the nmAbs completely prevented viremia in serum after challenge. Given that numerous antibodies have exceptional safety profiles in humans, the cocktail described here could be rapidly developed to protect uninfected pregnant women and their fetuses.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.