Scientific Publications

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

Introduction:
In Uganda, people living in fishing communities tend to engage in high-risk sexual activity which leads to unintended pregnancies that may end in abortions. Abortion has negative social, psychological, and medical impacts. We determined the frequency of abortion and its correlates among female fisher-folk along Lake Victoria in Uganda.

Methods:
A cross-sectional survey was conducted among women aged 15– 49 years from Kigungu and Nsazi fishing communities. Data were collected on socio-demographic characteristics, abortion, and family planning use. Associations between abortion and participant characteristics were assessed using logistic regression models.

Results:
Of the 713 women interviewed, 36, 5% were pregnant and 247, 34.6 % were using contraception. Majority (600, 84.2%) of those interviewed reported ever being pregnant. Approximately 45% of the pregnancies were un-intended while a third of those who had ever been pregnant (195, 32.5%) reported having aborted before. Slightly over a third (247, 34.6%) reported currently using or ever using family planning. Women aged 30+ years were more likely to abort compared to those aged 15-29 years (aOR: 2.7; 95% CI: 1.23-5.91). Women who had living children were less likely to abort compared to those who didn’t have any living child (aOR: 0.06; 95% CI: 0.01 – 0.17).

Conclusion:
The rate of abortion among female fisher-folk in Uganda is substantial. Family planning use is still low and unintended pregnancies are common. Abortion risk increased with the age of the mother. Continuous behavioral change communication and optimization of family planning use are recommended to reduce abortions.

The HIV Research for Prevention (HIVR4P) conference catalyzes knowledge sharing on biomedical HIV prevention interventions such as HIV vaccines, antibody infusions, pre-exposure prophylaxis, and microbicides in totality-from the molecular details and delivery formulations to the behavioral, social, and structural underpinnings. HIVR4P // Virtual was held over the course of 2 weeks on January 27-28 and February 3-4, 2021 as the coronavirus disease 2019 (COVID-19) pandemic continued to inflict unprecedented harm globally. The HIVR4P community came together with 1,802 researchers, care providers, policymakers, implementers, and advocates from 92 countries whose expertise spanned the breadth of the HIV prevention pipeline from preclinical to implementation. The program included 113 oral and 266 poster presentations. This article presents a brief summary of the conference highlights. Complete abstracts, webcasts, and daily rapporteur summaries may be found on the conference website (https://www.hivr4p.org/).

Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.

Forty years since the first report of the disease in the United States now known as HIV/AIDS, an efficacious HIV vaccine remains an elusive goal. However, the remarkable level of discovery, innovation and collaboration employed in pursuit of this goal has augmented the scientific insight and capabilities of vaccinology overall and benefitted vaccine development targeting other infectious diseases.

A promising vaccine fails to provide durable protection against infection and clinical malaria in infants, a key malaria vaccine target population, in a phase 2b clinical trial. The need for a highly effective vaccine against malaria remains as urgent as ever.

Most studies of HIV-1 transmission have focused on subtypes B and C. In this study, we determined the genomic sequences of the transmitted founder (TF) viruses from acutely infected individuals enrolled between 2005 and 2011 into IAVI protocol C in Rwanda and have compared these isolates to viruses from more recent (2016-2019) acute/early infections in three at risk populations - MSM, high risk women (HRW), and discordant couples (DC). For the Protocol C samples, we utilized near full-length single genome (NFLG) amplification to generate 288 HIV-1 amplicons from 26 acutely infected seroconverters (SC), while for the 21 recent seroconverter samples (13 from HRW, two from DC, and six from MSM), we PCR amplified overlapping half-genomes. Using PacBio SMRT technology combined with the MDPseq workflow, we performed multiplex sequencing to obtain high accuracy sequences for each amplicon. Phylogenetic analyses indicated that the majority of recent transmitted viruses from DC and HRW clustered within those of the earlier Protocol C cohort. However, five of six sequences from the MSM cohort branched together and were greater than 97% identical. Recombination analyses revealed a high frequency (6/26; 23%) of unique inter-subtype recombination in Protocol C with 19% AC and 4% CD recombinant viruses, which contrasted with only 6.5% of recombinants defined by sequencing of the gene previously. The frequency of recombinants was significantly higher (12/21; 57%) in the more recent isolates, although, the five related viruses from the MSM cohort had identical recombination break points. While major drug resistance mutations were absent from Protocol C viruses, 4/21 of recent isolates exhibited transmitted nevirapine resistance. These results demonstrate the ongoing evolution and increased prevalence of recombinant and drug resistant transmitted viruses in Rwanda and highlight the importance of defining NFLG sequences to fully understand the nature of TF viruses and in particular the prevalence of unique recombinant forms (URFs) in transmission cohorts.

Human immunodeficiency virus (HIV)-1-specific broadly neutralizing monoclonal antibodies are currently under development to treat and prevent HIV-1 infection. We performed a single-center, randomized, double-blind, dose-escalation, placebo-controlled trial of a single administration of the HIV-1 V3-glycan-specific antibody PGT121 at 3, 10 and 30 mg kg in HIV-uninfected adults and HIV-infected adults on antiretroviral therapy (ART), as well as a multicenter, open-label trial of one infusion of PGT121 at 30 mg kg in viremic HIV-infected adults not on ART (no. NCT02960581). The primary endpoints were safety and tolerability, pharmacokinetics (PK) and antiviral activity in viremic HIV-infected adults not on ART. The secondary endpoints were changes in anti-PGT121 antibody titers and CD4 T-cell count, and development of HIV-1 sequence variations associated with PGT121 resistance. Among 48 participants enrolled, no treatment-related serious adverse events, potential immune-mediated diseases or Grade 3 or higher adverse events were reported. The most common reactions among PGT121 recipients were intravenous/injection site tenderness, pain and headache. Absolute and relative CD4 T-cell counts did not change following PGT121 infusion in HIV-infected participants. Neutralizing anti-drug antibodies were not elicited. PGT121 reduced plasma HIV RNA levels by a median of 1.77 log in viremic participants, with a viral load nadir at a median of 8.5 days. Two individuals with low baseline viral loads experienced ART-free viral suppression for ≥168 days following antibody infusion, and rebound viruses in these individuals demonstrated full or partial PGT121 sensitivity. The trial met the prespecified endpoints. These data suggest that further investigation of the potential of antibody-based therapeutic strategies for long-term suppression of HIV is warranted, including in individuals off ART and with low viral load.

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184-186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.

Maternal mortality is still a challenge in Uganda, at 336 deaths per 100,000 live births, especially in rural hard to reach communities. Distance to a health facility influences maternal deaths. We explored women's mobility for maternal health, distances travelled for antenatal care (ANC) and childbirth among hard-to-reach Lake Victoria islands fishing communities (FCs) of Kalangala district, Uganda.

Acute retroviral syndrome (ARS) is associated with HIV-1 subtype and disease progression, but the underlying immunopathological pathways are poorly understood. We aimed to elucidate associations between innate immune responses during hyperacute HIV-1 infection (hAHI) and ARS.

Cytotoxic-T-Lymphocytes (CTL) are a critical arm of the immune response to viral infections. The activation and expansion of antigen specific CTL requires recognition of peptide antigens presented on class I major histocompatibility complex molecules (MHC-1) of infected cells. Methods to identify presented peptide antigens that do not rely on the pre-existence of antigen specific CTL are critical to the development of new vaccines. We infected activated CD4+ T cells with two HIV-1 Transmitted Founder (TF) isolates and used high-resolution mass spectrometry (MS) to identify HIV peptides bound on MHC-1. Using this approach, we identified 14 MHC-1 bound peptides from across the two TF isolates. Assessment of predicted binding thresholds revealed good association of the identified peptides to the shared HLA alleles between the HIV+ donors and the naïve PBMC sample with three peptides identified through peptide sequencing inducing a CD8 T-cell response (p<0.05). Direct infection of naïve CD4 cells by HIV transmitted founder isolates and sequencing of MHC-I presented peptides by HPLC-MS/MS enables identification of novel peptides that may be missed by alternative epitope mapping strategies and can provide valuable insight in to the first peptides presented by an HIV infected CD4 cell in the first few days post infection. This article is protected by copyright. All rights reserved.

Existing approaches to identifying predictive T-cell epitopes have traditionally utilized either 2-digit HLA super-families or more commonly utilizing autologous HLA alleles to facilitate the predictions. However, the use of these criteria may not consider the HLA representation within any target population. Here we propose a modification to concept of utilizing autologous HLA whereby subsets of individuals are selected for their specific HLA allele profiles and the representation they provide within a given population. Using this selective approach to HLA selection and the linkages to specific individuals may enable the design of more targeted experimental strategies. This article is protected by copyright. All rights reserved.