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. 2024 Jan 23;98(1):e0147823.
doi: 10.1128/jvi.01478-23. Epub 2023 Dec 12.

Development of LIBRA-seq for the guinea pig model system as a tool for the evaluation of antibody responses to multivalent HIV-1 vaccines

Matthew J Vukovich  1   2 , Nagarajan Raju  1   2 , Prudence Kgagudi  3 , Nelia P Manamela  3   4 , Alexandra A Abu-Shmais  1   2 , Kathryn R Gripenstraw  1 , Perry T Wasdin  1   5 , Xiaoying Shen  6 , Bridget Dwyer  7 , Jumana Akoad  7 , Rebecca M Lynch  7 , David C Montefiori  6 , Simone I Richardson  3   4 , Penny L Moore  3   4 , Ivelin S Georgiev  1   2   5   8   9   10   11
Affiliations

Development of LIBRA-seq for the guinea pig model system as a tool for the evaluation of antibody responses to multivalent HIV-1 vaccines

Matthew J Vukovich et al. J Virol. .

Abstract

Consistent elicitation of serum antibody responses that neutralize diverse clades of HIV-1 remains a primary goal of HIV-1 vaccine research. Prior work has defined key features of soluble HIV-1 Envelope (Env) immunogen cocktails that influence the neutralization breadth and potency of multivalent vaccine-elicited antibody responses including the number of Env strains in the regimen. We designed immunization groups that consisted of different numbers of SOSIP Env strains to be used in a cocktail immunization strategy: the smallest cocktail (group 2) consisted of a set of two Env strains, which were a subset of the three Env strains that made up group 3, which, in turn, were a subset of the six Env strains that made up group 4. Serum neutralizing titers were modestly broader in guinea pigs that were immunized with a cocktail of three Envs compared to cocktails of two and six, suggesting that multivalent Env immunization could provide a benefit but may be detrimental when the cocktail size is too large. We then adapted the LIBRA-seq platform for antibody discovery to be compatible with guinea pigs, and isolated several tier 2 neutralizing monoclonal antibodies. Three antibodies isolated from two separate guinea pigs were similar in their gene usage and CDR3s, establishing evidence for a guinea pig public clonotype elicited through vaccination. Taken together, this work investigated multivalent HIV-1 Env immunization strategies and provides a novel methodology for screening guinea pig B cell receptor antigen specificity at a high-throughput level using LIBRA-seq.IMPORTANCEMultivalent vaccination with soluble Env immunogens is at the forefront of HIV-1 vaccination strategies but little is known about the influence of the number of Env strains included in vaccine cocktails. Our results suggest that adding more strains is sometimes beneficial but may be detrimental when the number of strains is too high. In addition, we adapted the LIBRA-seq platform to be compatible with guinea pig samples and isolated several tier 2 neutralizing monoclonal antibodies, some of which share V and J gene usage and >70% CDR3 identity, thus establishing the existence of public clonotypes in guinea pigs elicited through vaccination.

Keywords: HIV-1; LIBRA-seq; guinea pig; immunogen; monoclonal antibodies; vaccine.

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Conflict of interest statement

M.J.V. and I.S.G. are listed as inventors on patents filed describing the antibodies discovered here. I.S.G. is listed as an inventor on the patent applications for the LIBRA-seq technology. I.S.G. is a co-founder of AbSeek Bio. I.S.G. has served as a consultant for Sanofi. The Georgiev laboratory at VUMC has received unrelated funding from Merck and Takeda Pharmaceuticals.

Figures

Fig 1
Fig 1
Vaccine groups were designed to evaluate the effect of including different numbers of Env strains in a cocktail immunization strategy. (A) Vaccine groups were composed of two strains (groups 1 and 2), three strains (group 3), or six strains (group 4). Group 1 consisted of two strains that have traditionally been used in the past. Envs in groups 2–4 were chosen to primarily assess the effect of having different numbers of Env strains included in the vaccine and were chosen by maximizing Env amino acid sequence diversity, as well as selecting strains with intact glycan shields and high sensitivity to all major bNAb classes. (B) Glycan occupancy at conserved glycosylation positions for each group is shown. Tan boxes indicate that all strains in a given group have a PNGS at the specified position. Pink boxes indicate a glycan hole, with the number denoting how many strains in the group are missing the specified glycan. Blue boxes indicate a glycan hole that is compensated for by a neighboring glycan, with the number denoting how many strains in the group have the compensatory glycan. Boxes with two colors indicate the presence of both missing and compensatory glycans at the specified position. (C) Vaccine strains are shown with the minimum IC50 values for each major bNAb class. Antibodies were grouped into bNAb classes based on epitopes targeted and other similarities such as angle of approach. (D) Guinea pigs were immunized with 100 µg total SOSIP.DS.644.sc immunogen at three intervals 4 weeks apart. Final bleeds and splenocytes were collected 4 weeks after the final dose.
Fig 2
Fig 2
Immunogen production and validation. (A) Immunogens displayed the expected size on SDS-PAGE. (B) Immunogens displayed expected FPLC SEC profiles. Dotted lines indicate fractions collected indicative of trimers. (C) Antigenicity of the immunogens was as expected measured by ELISA using a panel of standard antibodies including a CD4 binding site antibody (3BNC117), a quaternary-specific antibody (PGDM1400), as well as antibodies that recognize the open form of the trimer (F105 and 447–52d).
Fig 3
Fig 3
Serum ELISA binding and effector functions. (A) Sera from all time points were tested for binding against all strains of Env antigen in the vaccine regimen in an ELISA format. Final bleeds from all groups showed high binding to all Envs tested, whereas pre-bleed samples showed no binding. (B) Sera from all groups showed measurable ADCP responses to two strains of Env antigen. Fluorescent beads with immobilized streptavidin were coated with biotinylated Env trimer and subsequently mixed with sera from each guinea pig. THP-1 monocytes were then added and ADCP was quantified as the internalization or engulfment of the antigen-coated beads by THP-1 cells measured by flow cytometry. This internalization is indicated as an ADCP score. All groups were compared using a Kruskal-Wallis test and did not reach statistical significance for either antigen.
Fig 4
Fig 4
Serum neutralization data for vaccine groups. (A) Guinea pig sera were tested for neutralization against tier 2 HIV-1 pseudoviruses displaying Envs from vaccine strains (A–H) and the standard global panel (I–S) in the TZM-bl cell neutralization assay. Each row displays serum neutralization data from one guinea pig and each column represents neutralization data against a given strain of pseudovirus. ID50 values are reported as colors ranging from no neutralization detected (blue) to high potency neutralization (dark red). Detectable responses per animal were compared by a two-sided Wilcoxon test. There was a statistically significant difference between groups 2 and 3 (P = 0.006), with group 3 outperforming group 2. Groups 3 and 4 were comparable but trended toward group 3 having a broader median response (P = 0.2). Groups 2 and 4 were comparable (P = 0.6). There was a statistically significant difference between groups 1 and 3 (P = 0.004), with group 3 outperforming group 1. Raw ID50 values were subtracted from the ID50 values for the HIV-1 specificity control, murine leukemia virus. Responses that were equal to or greater than 20 after subtraction were considered positive. Arrows denote guinea pigs 109 (top) and 112 (bottom), from which splenocytes were used for monoclonal antibody isolation. (B) Comparison of breadth between groups as a function of the proportion of guinea pigs in each group that exhibit greater than or equal to that breadth denoted on the x-axis. The median, maximum, and minimum number of responses per group are shown to the right.
Fig 5
Fig 5
Development of LIBRA-seq with guinea pig B cells. (A) A flow panel was designed to isolate antigen-positive guinea pig B cells from splenocytes obtained from guinea pigs 109 and 112, chosen based on their broad serum neutralization profiles. Lymphocytes were gated out based on size, followed by live cells (data not shown). Cells with high IgG expression and negative IgM expression were then gated followed by antigen-positive B cells. (B) The LIBRA-seq workflow was adapted for use in guinea pigs by modifying BCR sequencing steps. Primers shown in red indicate guinea pig-specific reverse primers that anneal to the constant regions of the guinea pig IgG genes. These custom reverse primers were mixed with existing forward primers specific to sequences appended during single-cell sequencing using the Chromium Next GEM Single Cell V(D)J Reagent Kit. Following target enrichment, single-cell sequencing was carried out as described in the methods. The LIBRA-seq antigen screening panel is shown to the right and consists of 5 Envs, the RSC3 antigen, and an influenza HA CA/09 (negative control). Sequences of the primers used in guinea pig LIBRA-seq are shown.
Fig 6
Fig 6
Identification and validation of tier 2 neutralizing monoclonal antibodies from vaccinated guinea pigs using LIBRA-seq. (A) The LIBRA-seq output showing prioritized antibodies from guinea pig samples with predicted antigen specificities is shown. Each row consists of one B cell with the LIBRA-seq scores for each antigen, ranging from low (white) to high (blue), as well as an ELISA area under the curve with the same color scheme. Antibodies isolated from guinea pig 109 have a 109 before their number, and antibodies from guinea pig 112 have a 112 before their number. (B) Six neutralizing antibodies were identified from the positive hits. The virus strain is indicated by the title for each curve. (C) Neutralizing antibodies were tested against the vaccine panel and 9-strain global panel. IC50 values are displayed in μg/mL. Bolded numbers indicate neutralization was detected.
Fig 7
Fig 7
Guinea pig antibody gene usage and public clonotype analysis. (A) Antibody sequences were aligned to published guinea pig V genes. CDR3 sequences are shown along with CDR3 lengths. (B) Sequence analysis revealed a guinea pig public clonotype. Antibodies 109–5 (from guinea pig 109) along with antibodies 112–22 and 112–23 (from guinea pig 112) share the same variable and joining gene usage and have >70% CDR3 identity. Antibodies 112–7, 112–8, and 112–9 aligned to their inferred variable and joining germline genes are shown.
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