Immune escape mutations selected by neutralizing antibodies in natural HIV-1 infection can alter coreceptor usage repertoire of the transmitted/founder virus

The ability of HIV-1 to evade neutralizing antibodies (NAbs) in vivo is well demonstrated, but the impact of NAb escape mutations on HIV-1 phenotype other than immune escape itself has rarely been studied. Here, we show that immune escape mutations selected by V3-glycan specific NAbs in vivo can alter the coreceptor usage repertoire of the transmitted/founder (T/F) HIV-1. In a participant developed V3-glycan NAb response, naturally selected mutations at the V3 N301 and N332 glycan sites abrogated CCR8 usage while conferred APJ usage on the cognate T/F strain. Mutations at the N301 glycan also impaired CCR3 usage and partially compromised the efficiency in using CCR5, which could be fully restored by a single escape mutation at the N332 glycan site. Our study demonstrates the link between NAb escape and coreceptor usage alteration in natural HIV-1 infection and indicates that NAb response could drive virus entry tropism evolution in vivo.

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies

Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

Limited evidence for a relationship between HIV-1 glycan shield features in early infection and the development of neutralization breadth

Identifying if viral features present in acute HIV-1 infection predetermine the development of neutralization breadth is critical to vaccine design. Incorporating such features in vaccine antigens could initiate cross-reactive antibody responses that could sufficiently protect vaccinees from HIV-1 infection despite the uniqueness of each founder virus. To understand the relationship between Env determinants and the development of neutralization breadth, we focused on 197 individuals enrolled in two cohorts in Thailand and East Africa (RV144 and RV217) and followed since their diagnosis in acute or early HIV-1 infection. We analyzed the distribution of variable loop lengths and glycans as well as the predicted density of the glycan shield and compared these envelope features to the neutralization breadth data obtained three years after infection (n = 121). Our study revealed limited evidence for glycan shield features that associate with the development of neutralization breadth. While the glycan shield tended to be denser in participants who subsequently developed breadth, no significant relationship was found between the size of glycan holes and the development of neutralization breadth. The parallel analysis of 3,000 independent Env sequences showed no evidence of directional evolution of glycan shield features since the beginning of the epidemic. Together, our results highlight that glycan shield features in acute and early HIV-1 infection may not play a role determinant enough to dictate the development of neutralization breadth, and instead suggest that the glycan shield’s reactive properties that are associated with immune evasion may have a greater impact. IMPORTANCE A major goal of HIV-1 vaccine research is to design vaccine candidates that elicit potent broadly neutralizing antibodies (bnAbs). Different viral features have been associated with the development of broadly neutralizing antibodies, including the glycan shield on the surface of the HIV-1 Envelope (Env). Here, we analyzed data from two cohorts of individuals who were followed from early infection to several years after infection spanning multiple HIV-1 subtypes. We compared Env glycan features in HIV-1 sequences obtained in early infection to the potency and breadth of neutralizing antibodies measured one to three years after infection. We found limited evidence of glycan shield properties that associate with the development of neutralization breadth in these cohorts. These results may have important implications for antigen design in future vaccine strategies and emphasize that HIV-1 vaccines will need to rely on a complex set of properties to elicit neutralization breadth.

Mutational networks of escape from transmitted HIV-1 infection

Human immunodeficiency virus (HIV) is subject to immune selective pressure soon after it establishes infection at the founder stage. As an individual progresses from the founder to chronic stage of infection, immune pressure forces a history of mutations that are embedded in envelope sequences. Determining this pathway of coevolving mutations can assist in understanding what is different with the founder virus and the essential pathways it takes to maintain infection. We have combined operations research and bioinformatics methods to extract key networks of mutations that differentiate founder and chronic stages for 156 subtype B and 107 subtype C envelope (gp160) sequences. The chronic networks for both subtypes revealed strikingly different hub-and-spoke topologies compared to the less structured transmission networks. This suggests that the hub nodes are impacted by the immune response and the resulting loss of fitness is compensated by mutations at the spoke positions. The major hubs in the chronic C network occur at positions 12, 137 (within the N136 glycan), and 822, and at position 306 for subtype B. While both founder networks had a more heterogeneous connected network structure, interestingly founder B subnetworks around positions 640 and 837 preferentially contained CD4 and coreceptor binding domains. Finally, we observed a differential effect of glycosylation between founder and chronic subtype B where the latter had mutational pathways significantly driven by N-glycosylation. Our study provides insights into the mutational pathways HIV takes to evade the immune response, and presents features more likely to establish founder infection, valuable for effective vaccine design.

Deep Sequencing Reveals Central Nervous System Compartmentalization in Multiple Transmitted/Founder Virus Acute HIV-1 Infection

HIV-1 disseminates to a broad range of tissue compartments during acute HIV-1 infection (AHI). The central nervous system (CNS) can serve as an early and persistent site of viral replication, which poses a potential challenge for HIV-1 remission strategies that target the HIV reservoir. CNS compartmentalization is a key feature of HIV-1 neuropathogenesis. Thus far, the timing of how early CNS compartmentalization develops after infection is unknown. We examined whether HIV-1 transmitted/founder (T/F) viruses differ between CNS and blood during AHI using single-genome sequencing of envelope gene and further examined subregions in pol and env using next-generation sequencing in paired plasma and cerebrospinal fluid (CSF) from 18 individuals. Different proportions of mostly minor variants were found in six of the eight multiple T/F-infected individuals, indicating enrichment of some variants in CSF that may lead to significant compartmentalization in the later stages of infection. This study provides evidence for the first time that HIV-1 compartmentalization in the CNS can occur within days of HIV-1 exposure in multiple T/F infections. Further understanding of factors that determine enrichment of T/F variants in the CNS, as well as potential long-term implications of these findings for persistence of HIV-1 reservoirs and neurological impairment in HIV, is needed.

Distinct antibody-mediated selection in a narrow-source HIV-1 outbreak among Chinese former plasma donors

ABSTRACTThe HIV-1 envelope protein mutates rapidly to evade recognition and killing, and is a major target of the humoral immune response and in vaccine development. Identification of common antibody epitopes for vaccine development have been complicated by large variations on both virus (different infecting founder strains) and host genetic levels. We studied HIV-1 envelopegp120evolution in 12 Chinese former plasma donors infected with a purportedly single founder virus, with the aim of identifying common antibody epitopes under immune selection. We found five amino acid sites to be under significant positive selection in ≥50% of the patients, and 22 sites housing mutations consistent with antibody-mediated selection. Despite strong selection pressure, some sites housed a limited repertoire of amino acids. Structural modelling revealed that most sites were located on the exposed distal edge of the Gp120 trimer, whilst wholly invariant sites clustered within the centre of the protein complex. Four sites, flanking the V3 hypervariable loop of the Gp120, represent novel antibody epitopes that may be suitable as vaccine candidates.