Interaction dynamics between innate and adaptive immune cells responding to SARS-CoV-2 vaccination in non-human primates.

As SARS-CoV-2 variants continue evolving, testing updated vaccines in non-human primates remains important for guiding human clinical practice. To date, such studies have focused on antibody titers and antigen-specific B and T cell frequencies. Here, we extend our understanding by integrating innate and adaptive immune responses to mRNA-1273 vaccination in rhesus macaques. We sorted innate immune cells from a pre-vaccine time point, as well as innate immune cells and antigen-specific peripheral B and T cells two weeks after each of two vaccine doses and used single-cell sequencing to assess the transcriptomes and adaptive immune receptors of each cell. We show that a subset of S-specific T cells expresses cytokines critical for activating innate responses, with a concomitant increase in CCR5-expressing intermediate monocytes and a shift of natural killer cells to a more cytotoxic phenotype. The second vaccine dose, administered 4 weeks after the first, elicits an increase in circulating germinal center-like B cells 2 weeks later, which are more clonally expanded and enriched for epitopes in the receptor binding domain. Both doses stimulate inflammatory response genes associated with elevated antibody production. Overall, we provide a comprehensive picture of bidirectional signaling between innate and adaptive components of the immune system and suggest potential mechanisms for the enhanced response to secondary exposure.

Primary exposure to SARS-CoV-2 variants elicits convergent epitope specificities, immunoglobulin V gene usage and public B cell clones.

An important consequence of infection with a SARS-CoV-2 variant is protective humoral immunity against other variants. However, the basis for such cross-protection at the molecular level is incompletely understood. Here, we characterized the repertoire and epitope specificity of antibodies elicited by infection with the Beta, Gamma and WA1 ancestral variants and assessed their cross-reactivity to these and the more recent Delta and Omicron variants. We developed a method to obtain immunoglobulin sequences with concurrent rapid production and functional assessment of monoclonal antibodies from hundreds of single B cells sorted by flow cytometry. Infection with any variant elicited similar cross-binding antibody responses exhibiting a conserved hierarchy of epitope immunodominance. Furthermore, convergent V gene usage and similar public B cell clones were elicited regardless of infecting variant. These convergent responses despite antigenic variation may account for the continued efficacy of vaccines based on a single ancestral variant.

Primary exposure to SARS-CoV-2 variants elicits convergent epitope specificities, immunoglobulin V gene usage and public B cell clones.

An important consequence of infection with a SARS-CoV-2 variant is protective humoral immunity against other variants. The basis for such cross-protection at the molecular level is incompletely understood. Here we characterized the repertoire and epitope specificity of antibodies elicited by Beta, Gamma and ancestral variant infection and assessed their cross-reactivity to these and the more recent Delta and Omicron variants. We developed a high-throughput approach to obtain immunoglobulin sequences and produce monoclonal antibodies for functional assessment from single B cells. Infection with any variant elicited similar cross-binding antibody responses exhibiting a remarkably conserved hierarchy of epitope immunodominance. Furthermore, convergent V gene usage and similar public B cell clones were elicited regardless of infecting variant. These convergent responses despite antigenic variation may represent a general immunological principle that accounts for the continued efficacy of vaccines based on a single ancestral variant.

Safety and virologic impact of the IL-15 superagonist N-803 in people living with HIV: a phase 1 trial.

There is no cure for HIV infection, and lifelong antiretroviral therapy (ART) is required. N-803 is an IL-15 superagonist comprised of an N72D mutant IL-15 molecule attached to its alpha receptor and a human IgG1 fragment designed to increase IL-15 activity. Preclinical studies with both HIV and SIV suggest that the drug has potential to reduce virus reservoirs by activating virus from latency and enhancing effector function. We conducted a phase 1 study of N-803 ( NCT02191098 ) in people living with HIV, the primary objective of which was to assess the safety and tolerability of the drug, with an exploratory objective of assessing the impact on peripheral virus reservoirs. ART-suppressed individuals were enrolled into a dose-escalation study of N-803 in four different cohorts (0.3, 1.0, 3.0 and 6.0 mcg kg-1). Each cohort received three doses total, separated by at least 1 week. We enrolled 16 individuals, of whom 11 completed all three doses. The maximum tolerated dose was 6.0 mcg kg-1. The primary clinical adverse events (AEs) reported were injection site rash and adenopathy, and four participants experienced a grade 1 or grade 2 QTc prolongation. No significant laboratory AEs attributable to N-803 were observed. In exploratory analyses, N-803 was associated with proliferation and/or activation of CD4+ and CD8+ T cells and natural killer cells that peaked at 4 d after dosing. IFN-γ, IP-10, MCP-1 and IL-15 increased during treatment. HIV transcription in memory CD4 T cells and intact proviral DNA initially increased after N-803 treatment; however, there was a small but significant decrease in the frequency of peripheral blood mononuclear cells with an inducible HIV provirus that persisted for up to 6 months after therapy. These data suggest that N-803 administration in ART-suppressed people living with HIV is safe and that larger clinical trials are needed to further investigate the effects of N-803 on HIV reservoirs.

Translocated microbiome composition determines immunological outcome in treated HIV infection.

The impact of the microbiome on HIV disease is widely acknowledged although the mechanisms downstream of fluctuations in microbial composition remain speculative. We detected rapid, dynamic changes in translocated microbial constituents during two years after cART initiation. An unbiased systems biology approach revealed two distinct pathways driven by changes in the abundance ratio of Serratia to other bacterial genera. Increased CD4 T cell numbers over the first year were associated with high Serratia abundance, pro-inflammatory innate cytokines, and metabolites that drive Th17 gene expression signatures and restoration of mucosal integrity. Subsequently, decreased Serratia abundance and downregulation of innate cytokines allowed re-establishment of systemic T cell homeostasis promoting restoration of Th1 and Th2 gene expression signatures. Analyses of three other geographically distinct cohorts of treated HIV infection established a more generalized principle that changes in diversity and composition of translocated microbial species influence systemic inflammation and consequently CD4 T cell recovery.

Pre-existing Immunity to Japanese Encephalitis Virus Alters CD4 T Cell Responses to Zika Virus Inactivated Vaccine.

The epidemic spread of Zika virus (ZIKV), associated with devastating neurologic syndromes, has driven the development of multiple ZIKV vaccines candidates. An effective vaccine should induce ZIKV-specific T cell responses, which are shown to improve the establishment of humoral immunity and contribute to viral clearance. Here we investigated how previous immunization against Japanese encephalitis virus (JEV) and yellow fever virus (YFV) influences T cell responses elicited by a Zika purified-inactivated virus (ZPIV) vaccine. We demonstrate that three doses of ZPIV vaccine elicited robust CD4 T cell responses to ZIKV structural proteins, while ZIKV-specific CD4 T cells in pre-immunized individuals with JEV vaccine, but not YFV vaccine, were more durable and directed predominantly toward conserved epitopes, which elicited Th1 and Th2 cytokine production. In addition, T cell receptor repertoire analysis revealed preferential expansion of cross-reactive clonotypes between JEV and ZIKV, suggesting that pre-existing immunity against JEV may prime the establishment of stronger CD4 T cell responses to ZPIV vaccination. These CD4 T cell responses correlated with titers of ZIKV-neutralizing antibodies in the JEV pre-vaccinated group, but not in flavivirus-naïve or YFV pre-vaccinated individuals, suggesting a stronger contribution of CD4 T cells in the generation of neutralizing antibodies in the context of JEV-ZIKV cross-reactivity.

An initiative of cooperation in Zika virus research: the experience of the ZIKABRA study in Brazil.

BACKGROUND: The Zika virus outbreak has triggered a set of local and global actions for a rapid, effective, and timely public health response. A World Health Organization (WHO) initiative, supported by the Department of Chronic Condition Diseases and Sexually Transmitted Infections (DCCI) of the Health Surveillance Secretariat (SVS), Brazil Ministry of Health (MoH) and other public health funders, resulted in the start of the "Study on the persistence of Zika virus in body fluids of patients with ZIKV infection in Brazil - ZIKABRA study". The ZIKABRA study was designed to increase understanding of how long ZIKV persists in bodily fluids and informing best measures to prevent its transmission. Data collection began in July 2017 and the last follow up visit occurred in 06/26/2020. METHODS: A framework for the ZIKABRA Cooperation initiative is provided through a description and analysis of the mechanisms, strategies and the ethos that have guided the models of international governance and technical cooperation in health for scientific exchange in the context of a public health emergency. Among the methodological strategies, we included a review of the legal documents that supported the ZIKABRA Cooperation; weekly documents produced in the meetings and working sessions; technical reports; memorandum of understanding and the research protocol. CONCLUSION: We highlight the importance of working in cooperation between different institutional actors to achieve more significant results than that obtained by each group working in isolation. In addition, we point out the advantages of training activities, ongoing supervision, the construction of local installed research capacity, training academic and non-academic human resources, improvement of laboratory equipment, knowledge transfer and the availability of the ZIKABRA study protocol for development of similar studies, favoring the collective construction of knowledge to provide public health emergency responses. Strategy harmonization; human resources and health services; timing and recruiting particularities and processing institutional clearance in the different sites can be mentioned as challenges in this type of initiative.

CTL Clonotypes with Higher TCR Affinity Have Better Ability to Reduce the HIV Latent Reservoir.

The success of the shock and kill strategy for the HIV cure depends both on the reactivation of the latent reservoir and on the ability of the immune system to eliminate infected cells. As latency reversal alone has not shown any impact in the size of the latent reservoir, ensuring that effector CTLs are able to recognize and kill HIV-infected cells could contribute to reservoir reduction. In this study, we investigated which functional aspects of human CTLs are associated with a better capacity to kill HIV-infected CD4+ T cells. We isolated Gag- and Nef-specific CTL clones with different TCR sequences from the PBMC of donors in acute and chronic infection. High-affinity clonotypes that showed IFN-γ production preserved even when the CD8 coreceptor was blocked, and clones with high Ag sensitivity exhibited higher efficiency at reducing the latent reservoir. Although intrinsic cytotoxic capacity did not differ according to TCR affinity, clonotypes with high TCR affinity showed a better ability to kill HIV-infected CD4+ T cells obtained from in vivo-infected PBMC and subjected to viral reactivation. Strategies aiming to specifically boost and maintain long-living memory CTLs with high TCR affinity in vivo prior to latency-reversing treatment might improve the efficacy of the shock and kill approach to reduce the latent reservoir.

Resistance to the Tat Inhibitor Didehydro-Cortistatin A Is Mediated by Heightened Basal HIV-1 Transcription.

Human immunodeficiency virus type 1 (HIV-1) Tat binds the viral RNA structure transactivation-responsive element (TAR) and recruits transcriptional cofactors, amplifying viral mRNA expression. The Tat inhibitor didehydro-cortistatin A (dCA) promotes a state of persistent latency, refractory to viral reactivation. Here we investigated mechanisms of HIV-1 resistance to dCA in vitro Mutations in Tat and TAR were not identified, consistent with the high level of conservation of these elements. Instead, viruses resistant to dCA developed higher Tat-independent basal transcription. We identified a combination of mutations in the HIV-1 promoter that increased basal transcriptional activity and modifications in viral Nef and Vpr proteins that increased NF-κB activity. Importantly, these variants are unlikely to enter latency due to accrued transcriptional fitness and loss of sensitivity to Tat feedback loop regulation. Furthermore, cells infected with these variants become more susceptible to cytopathic effects and immune-mediated clearance. This is the first report of viral escape to a Tat inhibitor resulting in heightened Tat-independent activity, all while maintaining wild-type Tat and TAR.IMPORTANCE HIV-1 Tat enhances viral RNA transcription by binding to TAR and recruiting activating factors. Tat enhances its own transcription via a positive-feedback loop. Didehydro-cortistatin A (dCA) is a potent Tat inhibitor, reducing HIV-1 transcription and preventing viral rebound. dCA activity demonstrates the potential of the "block-and-lock" functional cure approaches. We investigated the viral genetic barrier to dCA resistance in vitro While mutations in Tat and TAR were not identified, mutations in the promoter and in the Nef and Vpr proteins promoted high Tat-independent activity. Promoter mutations increased the basal transcription, while Nef and Vpr mutations increased NF-κB nuclear translocation. This heightened transcriptional activity renders CD4+ T cells infected with these viruses more susceptible to cytotoxic T cell-mediated killing and to cell death by cytopathic effects. Results provide insights on drug resistance to a novel class of antiretrovirals and reveal novel aspects of viral transcriptional regulation.

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.

T Cell Immunity and Zika Virus Vaccine Development.

The recent Zika virus (ZIKV) epidemic has created an urgent need for a safe and effective vaccine. There is still a dearth of knowledge about ZIKV immunity, but years of investigation into the immunobiology of other flaviviruses has helped to accelerate the development of a ZIKV vaccine. Although the humoral immune response generates the primary correlate of protection from disease, robust T cell responses could enhance ZIKV vaccine efficacy. Additionally, pre-existing immunity to related flaviviruses could generate cross-reactive T cells that may affect immune responses upon vaccination. In this review, we summarize the key discoveries in the area of flavivirus T cell immunity and postulate on how these findings can inform ZIKV vaccine strategies for inducing protective immunity.

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.

Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil.

BACKGROUND: Zika virus (ZIKV) is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barré syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications, prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Nine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak. CONCLUSIONS/SIGNIFICANCE: The detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological relevance of this finding, regarding the contribution of alternative non-vectorial ZIKV transmission routes, needs further investigation.

Vaccine-Induced Simian Immunodeficiency Virus-Specific CD8+ T-Cell Responses Focused on a Single Nef Epitope Select for Escape Variants Shortly after Infection.

UNLABELLED: Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed "elite controllers" [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08(+) animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08(+) macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8(+) T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8(+) T-cell response would facilitate the development of elite control in Mamu-B*08(+) animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08(+) animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8(+) T cells. These vaccine-induced effector memory CD8(+) T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8(+) T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08(+) macaques. IMPORTANCE: Since elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8(+) T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08(+) rhesus macaques­a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8(+) T-cell response targeting the conserved "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08(+) monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8(+) T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8(+) T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection.

Improved genetic stability of recombinant yellow fever 17D virus expressing a lentiviral Gag gene fragment.

We have previously designed a method to construct viable recombinant Yellow Fever (YF) 17D viruses expressing heterologous polypeptides including part of the Simian Immunodeficiency Virus (SIV) Gag protein. However, the expressed region, encompassing amino acid residues from 45 to 269, was genetically unstable. In this study, we improved the genetic stability of this recombinant YF 17D virus by introducing mutations in the IRES element localized at the 5' end of the SIV gag gene. The new stable recombinant virus elicited adaptive immune responses similar to those induced by the original recombinant virus. It is, therefore, possible to increase recombinant stability by removing functional motifs from the insert that may have deleterious effects on recombinant YF viral fitness.