Approaches to modulate vaccine-induced antibody responses

Most successful licensed vaccines mediate protection by inducing antibody responses capable of protecting the individual from infection or disease. Despite the availability of many effective vaccines, we lack knowledge about fundamental aspects of B cell immunity and how we respond to complex real-world protein antigens. HIV-1 is a particularly difficult vaccine target since it has evolved multiple strategies to evade host antibody responses, such as conformational and glycan shielding. A consequence of this effective shielding is that the most immunogenic determinants of the envelope glycoprotein (Env) spike are those that the virus can readily mutate, which give rise to strain-specific antibodies. The conserved determinants of Env, which are targets for broadly neutralizing antibodies, tend to be less immunogenic. Another challenge has been the production of recombinant immunogens that mimic the functional HIV-1 Env spike but recent developments have improved this situation. In this thesis, I studied Env-specific immune responses induced by immunization with recombinant Env trimers to broaden our knowledge about B cell responses against this complex protein antigen. In brief, I investigated basic B cell questions using HIV-1 Env as a model antigen in a series of mouse immunogenicity studies. In Paper I, we asked whether or not there is a competition between B cells that recognize distal epitopes on HIV-1 Env. The variable region 3 (V3) of Env is highly immunogenic. We therefore masked the V3 region with N-linked glycans to study if the elicited response would be re-distributed to other more conserved determinants on Env. The results indicated that there was no competition between B cells. Instead, we observed a total decrease in the antibody responses when the V3 region was shielded. Next, in Paper II, we manipulated the naïve B cell repertoire by pre-treating mice with recombinant BLyS to rescue additional B cells into the mature naïve B cell pool. The results showed that BLyS treatment expanded the naïve B cell population and resulted in improved neutralization capacity after Env immunization. In Paper III, we established protocols that would allow characterization of vaccine-induced antibody specificities. We describe staining protocols for Env-specific memory and germinal center B cells in mice. Finally, in Paper IV, we evaluated the effect of chemical cross-linking of HIV-1 Env trimers for the elicited antibody response after immunization. The results indicated a Th2 shift in the serum antibody isotype response compared to the response elicited in mice immunized with unfixed trimers. In conclusion, the purpose of this thesis has been to evaluate strategies to manipulate or redirect the antibody response induced by recombinant HIV-1 Env immunogens. The results obtained in this thesis gave insights into several fundamental B cell questions of relevance to the vaccine field and to our understanding of basic B cell immunology

Env-Specific Antibodies in Chronic Infection versus in Vaccination

Antibodies are central in vaccine-mediated protection. For HIV-1, a pathogen that displays extreme antigenic variability, B cell responses against conserved determinants of the envelope glycoproteins (Env) are likely required to achieve broadly protective vaccine-induced responses. To understand antibodies in chronic infection, where broad serum neutralizing activity is observed in a subset of individuals, monoclonal antibodies mediating this activity have been isolated. Studies of their maturation pathways reveal that years of co-evolution between the virus and the adaptive immune response are required for such responses to arise. Furthermore, they do so in subjects who display alterations of their B cell subsets caused by the chronic infection, conditions that are distinctly different from those in healthy hosts. So far, broadly neutralizing antibody responses were not induced by vaccination in primates or small animals with natural B cell repertoires. An increased focus on the development vaccine-induced responses in healthy subjects is therefore needed to delineate how the immune system recognizes different forms of HIV-1 Env and to optimize approaches to stimulate antibody responses against relevant neutralizing antibody epitopes. In this review, we describe aspects of Env-directed antibody responses that differ between chronic HIV-1 infection and subunit vaccination for an increased appreciation of these differences; and we highlight the need for an improved understanding of vaccine-induced B cell responses to complex glycoproteins such as Env, in healthy subjects

HIV-1 Env-Specific Memory and Germinal Center B Cells in C57BL/6 Mice

Continued efforts to define the immunogenic properties of the HIV-1 envelope glycoproteins (Env) are needed to elicit effective antibody (Ab) responses by vaccination. HIV-1 is a highly neutralization-resistant virus due to conformational and glycan shielding of conserved Ab determinants on the virus spike. Elicitation of broadly neutralizing Abs that bind poorly accessible epitope regions on Env is therefore extremely challenging and will likely require selective targeting of specific sub-determinants. To evaluate such approaches there is a pressing need for in vivo studies in both large and small animals, including mice. Currently, most mouse immunization studies are performed in the BALB/c strain; however, the C57BL/6 strain offers improved possibilities for mechanistic studies due to the availability of numerous knock-out strains on this genetic background. Here, we compared Env immunogenicity in BALB/c and C57BL/6 mice and found that the magnitude of the antigen-specific response was somewhat lower in C57BL/6 than in BALB/c mice by ELISA but not significantly different by B cell ELISpot measurements. We then established protocols for the isolation of single Env-specific memory B cells and germinal center (GC) B cells from immunized C57BL/6 mice to facilitate future studies of the elicited response at the monoclonal Ab level. We propose that these protocols can be used to gain an improved understanding of the early recruitment of Env-specific B cells to the GC as well as the archiving of such responses in the memory B cell pool following immunization

Glutaraldehyde Cross-linking of HIV-1 Env Trimers Skews the Antibody Subclass Response in Mice

Well-ordered soluble HIV-1 envelope glycoprotein (Env) spike mimetics such as Native Flexibly Linked (NFL) trimers display high homogeneity, desired antigenicity, and high in vitro stability compared to previous generation soluble HIV-1 Env trimers. Glutaraldehyde (GLA) cross-linking was shown to further increase the thermostability of clade C 16055 NFL trimers and enhance the induction of tier 2 autologous neutralizing antibodies in guinea pigs. Here, we investigated if GLA fixation affected other aspects of the Env-specific immune response by performing a comparative immunogenicity study in C57BL/6 mice with non-fixed and GLA-fixed 16055 NFL trimers administered in AbISCO-100 adjuvant. We detected lower Env-specific binding antibody titers and increased skewing toward Th2 responses in mice immunized with GLA-fixed trimers compared to mice immunized with unfixed trimers, as shown by a higher Env-specific IgG1:IgG2b antibody subclass ratio. These results suggest that the presence of GLA adducts on Env influences the quality of the induced antibody response

HIV-1 Receptor Binding Site-Directed Antibodies Using a VH1-2 Gene Segment Orthologue Are Activated by Env Trimer Immunization

<div><p>Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01.</p></div

Genetic analysis of the isolated VH1.23-using CD4bs-specific MAbs.

<p>(A) Sequence alignments of the isolated NHP VH1.23-using MAbs. The framework regions (FR) are boxed in gray and the amino acid differences compared to the germline VH1.23 sequence are highlighted in yellow. Depicted below the MAb sequences is the newly discovered VH1.23*02 germline allele. (B) Alignments of the GE356 heavy and light chain sequences with the assigned germline sequences. (C) Summary of the genetic characteristics of the VH1.23 vaccine-induced MAbs.</p

Representation of the GE356 Fab crystal structure and paratope mapping analysis.

<p>(A) The GE356 heavy chain is shown in dark blue and light chain in light blue. The HCDR and LCDR loops are colored as follows: CDR1s (green), CDR2s (orange), CDR3s (magenta). (B) Effects of alanine substitutions in the CDRs of the heavy and light chain of GE356 on YU2 and SF162 gp120 binding (left) by ELISA and neutralization capacity (HXBc2 and SF162, right), as measured using the TZM-bl pseudovirus assay, compared to WT GE356. Residues are numbered based on the Kabat numbering system. EC₅₀ (Ab concentration for half-maximal binding) and IC₅₀ (Ab concentration for 50% neutralization) values are shown. The color-coding reflects fold-decrease in binding or of neutralization activity as compared to WT, in red (>50× WT), dark orange (20–50× WT), light orange (10–20× WT) and yellow (5–10× WT). HCDR3 A93 and LCDR3 A95b were not included in the analysis as there were alanines in these positions in the WT sequence.</p

Isolation of HIV-1 gp120 CD4bs-specific vaccine-induced NHP memory B cells, NHP MAbs and their binding specificities.

<p>(A) Structural representation of the TriMut and TriMut368/370 probes that were used for sorting CD4bs-specific memory B cells. The key mutations for TriMut (I423M, N425K and G431E) are highlighted in hot pink. The additional TriMut368/370 mutations (D368R and E370F) used for negative selection are highlighted in green and the CD4 footprint is in yellow. The inserted table shows the binding profile of the probes with a panel of CD4bs antibodies and CD4-Ig. Relative binding to the probes was based on the OD450 values at the highest concentration of protein (10 µg/ml) and EC₅₀ values: OD450≥3.0 and EC₅₀≤0.10 = ++++; OD450≥3.0 and EC₅₀>0.10 = +++; 2.0≤OD450<3.0 = ++; 0.2≤OD450<2.0 = +; OD450<0.2 = −. (B) Details describing the flow cytometric sorting of CD4bs-directed memory B cells using the TriMut and TriMut368/370 probes. Gating strategy for sorting IgG⁺ and CD4bs-specific memory B cells; CD20⁺, IgG⁺, CD27⁺, CD3⁻, CD8⁻, CD14⁻, IgM⁻, TriMut and TriMut-368/370 were sorted at single cell density. (C) Four VH1.23-using MAbs (GE331, GE356, GE443 and GE453) were characterized for their capacity to bind trimeric gp140-F+/−D368R and TriMut+/−368/370 by ELISA. Titration curves are shown as Log₁₀ dilutions (µg/ml). All antibodies were isolated from the post 2 immunization time point.</p

NHP VH1 gene segment usage.

<p>(A) Phylogenetic representation of known rhesus macaque VH germline sequences. The VH1 family is boxed and shown in a magnified view to highlight the VH1 rhesus macaque germline sequences in relation to the human VH1-2*02 germline sequence (red). (B) Sequence alignments of all known human VH1-2 alleles are shown in the top panel. Framework (FR) regions are shaded in gray with the amino acid differences between the alleles highlighted in blue. Sequence alignments of the human VH1-2*02 germline sequence with the known rhesus VH1 germline sequences are shown in the lower panel. Differences between human VH1-2*02 and the rhesus germline sequences are highlighted in orange and the percentage nucleic acid (NA) and amino acid (AA) homologies are indicated to the right after each germline sequence. The W50/N58/R71 motif, which is typically present in VH1-2*02-using bNAbs, is highlighted by the vertical red boxes. (C) Pie-chart indicating the family usage of 179 VH sequences isolated from animal F128. Sequence “slices” belonging to the VH1 family are boxed and highlighted in different shades of blue. The percentage of sequences annotated to a given germline gene segment within the VH1 family is indicated in parentheses in the magnified view to the right.</p