A VRC13-like bNAb response is associated with complex escape pathways in HIV-1 envelope.

The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N-linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens.IMPORTANCEThe pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope (env). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.

Heterotypic responses against nsp12/nsp13 from prior SARS-CoV-2 infection associates with lower subsequent endemic coronavirus incidence.

Immune responses from prior SARS-CoV-2 infection and COVID-19 vaccination do not prevent re-infections and may not protect against future novel coronaviruses (CoVs). We examined the incidence of and immune differences against human endemic CoVs (eCoV) as a proxy for response against future emerging CoVs. Assessment was among those with known SARS-CoV-2 infection, COVID-19 vaccination but no documented SARS-CoV-2 infection, or neither exposure. Retrospective cohort analyses suggest that prior SARS-CoV-2 infection, but not COVID-19 vaccination alone, protects against subsequent symptomatic eCoV infection. CD8+ T cell responses to the non-structural eCoV proteins, nsp12 and nsp13, were significantly higher in individuals with previous SARS-CoV-2 infection as compared to the other groups. The three groups had similar cellular responses against the eCoV spike and nucleocapsid, and those with prior spike exposure had lower eCoV-directed neutralizing antibodies. Incorporation of non-structural viral antigens in a future pan-CoV vaccine may improve protection against future heterologous CoV infections.

Identification of Genetically Related HCV Infections Among Self-Described Injecting Partnerships.

BACKGROUND: The current opioid epidemic across the United States has fueled a surge in the rate of new hepatitis C virus (HCV) infections among young persons who inject drugs (PWIDs). Paramount to interrupting transmission is targeting these high-risk populations and understanding the underlying network structures facilitating transmission within these communities. METHODS: Deep sequencing data were obtained for 52 participants from 32 injecting partnerships enrolled in the U-Find-Out (UFO) Partner Study, which is a prospective study of self-described injecting dyad partnerships from a large community-based study of HCV infection in young adult PWIDs from San Francisco. Phylogenetically linked transmission events were identified using traditional genetic-distance measures and viral deep sequence phylogenies reconstructed to determine the statistical support of inferences and the direction of transmission within partnerships. RESULTS: Using deep sequencing data, we found that 12 of 32 partnerships were genetically similar and clustered. Three additional phylogenetic clusters were found describing novel putative transmission links outside of the injecting relationship. Transmission direction was inferred correctly for 5 partnerships with the incorrect transmission direction inferred in more than 50% of cases. Notably, we observed that phylogenetic linkage was most often associated with a lower number of network partners and involvement in a sexual relationship. CONCLUSIONS: Deep sequencing of HCV among self-described injecting partnerships demonstrates that the majority of transmission events originate from outside of the injecting partnership. Furthermore, these findings caution that phylogenetic methods may be unable to routinely infer the direction of transmission among PWIDs especially when transmission events occur in rapid succession within high-risk networks.

Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection Associates With Unstable Housing and Occurs in the Presence of Antibodies.

BACKGROUND: The factors associated with severe acute respiratory coronavirus 2 (SARS-CoV-2) reinfection remain poorly defined. METHODS: We identified patients with SARS-CoV-2 infection and at least 1 repeat reverse transcription polymerase chain reaction result a minimum of 90 days after the initial positive test and before 21 January 2021. Those with a repeat positive test were deemed to have reinfection (n = 75), and those with only negative tests were classified as convalescents (n = 1594). Demographics, coronavirus disease 2019 (COVID-19) severity, and treatment histories were obtained from the Boston Medical Center electronic medical record. Humoral responses were analyzed using SARS-CoV-2-specific enzyme-linked immunosorbent assays and pseudovirus neutralizations in a subset of reinfection (n = 16) and convalescent samples (n = 32). Univariate, multivariate, and time to event analyses were used to identify associations. RESULTS: Individuals with reinfection had more frequent testing at shorter intervals compared with the convalescents. Unstable housing was associated with more than 2-fold greater chance of reinfection. Preexisting comorbidities and COVID-19 severity after the initial infection were not associated with reinfection. SARS-CoV-2 immunoglobulin G levels and pseudovirus neutralization were not different within the early weeks after primary infection and at a timepoint at least 90 days later in the 2 groups. In the convalescents, but not in those with reinfection, the late as compared with early humoral responses were significantly higher. CONCLUSIONS: Reinfection associates with unstable housing, which is likely a marker for virus exposure, and reinfection occurs in the presence of SARS-CoV-2 antibodies.

Family matters for coronavirus disease and vaccines.

The increasing frequency of pathogenic coronaviruses in the human population has raised public health concerns about possible future pandemics. It is critical to understand whether immune responses to the current circulating coronaviruses provide protection against related viruses or those that may emerge in the future. In this issue of the JCI, Dangi, Palacio, and co-authors detail the extent of coronavirus cross-protection following both vaccination and natural infection and ultimately used murine models to highlight the mechanism behind this heterotypic immunity. This study provides insight into the possibility of a pan-coronavirus vaccine that could protect humans against future coronavirus outbreaks.

Differentiation of exhausted CD8+ T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory.

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8+ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory.

Hepatitis C Virus Transmission Clusters in Public Health and Correctional Settings, Wisconsin, USA, 2016-20171.

Ending the hepatitis C virus (HCV) epidemic requires stopping transmission among networks of persons who inject drugs. Identifying transmission networks by using genomic epidemiology may inform community responses that can quickly interrupt transmission. We retrospectively identified HCV RNA-positive specimens corresponding to 459 persons in settings that use the state laboratory, including correctional facilities and syringe services programs, in Wisconsin, USA, during 2016-2017. We conducted next-generation sequencing of HCV and analyzed it for phylogenetic linkage by using the Centers for Disease Control and Prevention Global Hepatitis Outbreak Surveillance Technology platform. Analysis showed that 126 persons were linked across 42 clusters. Phylogenetic clustering was higher in rural communities and associated with female sex and younger age among rural residents. These data highlight that HCV transmission could be reduced by expanding molecular-based surveillance strategies to rural communities affected by the opioid crisis.

Role of HCV Viremia in Corroborated HCV Transmission Events Within Young Adult Injecting Partnerships.

BACKGROUND: Hepatitis C virus (HCV), a major cause of morbidity and mortality, is common and rising among young persons who inject drugs (PWID). Reducing the level of viremia may be an intervention, yet the impact of viremia on HCV transmission is unknown. METHODS: We conducted a prospective study of injecting partnerships (Partner Study) of young adult (age < 30 years) PWID within the UFO Study, which enrolled those at risk for HCV or with seronegative viremic infection and up to 3 HCV RNA-positive regular injecting partners. We examined the level of HCV viremia and stage of infection in the HCV-positive partner in regression analyses of HCV transmission events that were corroborated via HCV phylogenetic linkage analyses. RESULTS: We enrolled 69 at-risk/acutely infected PWID. There were 25 new HCV infections (incidence rate, 35.9 per 100 person-years; 95% confidence interval [CI], 24.3-53.2 per 100 person-years); 12/25 (48%) were phylogenetically linked to at least 1 partner. We found no association between the infected partner's quantitative level of HCV viremia and likely transmission in multivariate analyses (adjusted odds ratio [AOR], 0.90; 95% confidence interval [CI], 0.55-1.46); however, seronegative viremic infection in the infected partner was associated with increased transmission (AOR, 28.02; 95% CI, 5.61-139.95). CONCLUSIONS: The HCV viremia level was not associated with increased odds of transmission, yet acute HCV infection (seronegative viremic) was. Explanations include high-risk behavior during acute infection or missed fluctuations in viremia during acute infection. Both point to the need for frequent testing to detect new infection and attempt to prevent onward transmission.

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.

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.

High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease.

BACKGROUND & AIMS: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. METHODS: To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. RESULTS: HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. CONCLUSIONS: We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization. LAY SUMMARY: HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.

Differences in the Selection Bottleneck between Modes of Sexual Transmission Influence the Genetic Composition of the HIV-1 Founder Virus.

Due to the stringent population bottleneck that occurs during sexual HIV-1 transmission, systemic infection is typically established by a limited number of founder viruses. Elucidation of the precise forces influencing the selection of founder viruses may reveal key vulnerabilities that could aid in the development of a vaccine or other clinical interventions. Here, we utilize deep sequencing data and apply a genetic distance-based method to investigate whether the mode of sexual transmission shapes the nascent founder viral genome. Analysis of 74 acute and early HIV-1 infected subjects revealed that 83% of men who have sex with men (MSM) exhibit a single founder virus, levels similar to those previously observed in heterosexual (HSX) transmission. In a metadata analysis of a total of 354 subjects, including HSX, MSM and injecting drug users (IDU), we also observed no significant differences in the frequency of single founder virus infections between HSX and MSM transmissions. However, comparison of HIV-1 envelope sequences revealed that HSX founder viruses exhibited a greater number of codon sites under positive selection, as well as stronger transmission indices possibly reflective of higher fitness variants. Moreover, specific genetic "signatures" within MSM and HSX founder viruses were identified, with single polymorphisms within gp41 enriched among HSX viruses while more complex patterns, including clustered polymorphisms surrounding the CD4 binding site, were enriched in MSM viruses. While our findings do not support an influence of the mode of sexual transmission on the number of founder viruses, they do demonstrate that there are marked differences in the selection bottleneck that can significantly shape their genetic composition. This study illustrates the complex dynamics of the transmission bottleneck and reveals that distinct genetic bottleneck processes exist dependent upon the mode of HIV-1 transmission.

Deep sequencing of hepatitis C virus reveals genetic compartmentalization in cerebrospinal fluid from cognitively impaired patients.

BACKGROUND & AIMS: Hepatitis C virus (HCV) causes neuropsychiatric impairment and fatigue with recent studies suggesting HCV invasion of the central nervous system (CNS). Our previous finding that endothelial cells from the blood-brain barrier support HCV infection warrants further investigation to elucidate whether the CNS can serve as a reservoir for independent HCV evolution. METHODS: Cerebrospinal fluid (CSF) and plasma from six HCV-infected patients without liver disease or co-morbidities together with plasma from six healthy subjects were profiled for markers of immune activation and viral quasispecies measured by deep sequencing. Unsupervised data analyses were used to identify any associations between cytokine activation markers and clinical outcomes. RESULTS: Four of six HCV-infected patients showed significant evidence of cognitive dysfunction and fatigue. Deep sequencing revealed independent viral evolution within the CNS of two cognitively impaired patients. Principal component analysis of peripheral cytokines demonstrated that individuals without cognitive impairment clustered together while a distinct cytokine pattern emerged with patients exhibiting cognitive dysfunction and fatigue. CONCLUSIONS: Deep sequencing demonstrated unique viral variants in the CSF of two cognitively impaired patients consistent with CNS replication or sequestration. Meanwhile, compartmentalization was absent in infected patients with no neurocognitive impairment. Examination of cytokine profiles in HCV-infected patients with cognitive dysfunction revealed elevated peripheral cytokine levels resulting in a distinct cytokine profile that may be related to cognitive impairment or viral penetration into the CNS. Further studies to determine the significance of unique HCV variants within the CNS are warranted.

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.