HIV-1 receptor binding site-directed antibodies using a VH1-2 gene segment orthologue are activated by Env trimer immunization.

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.

Slc15a4 function is required for intact class switch recombination to IgG2c in response to TLR9 stimulation.

Signalling through Toll-like receptors (TLRs) by endogenous components of viruses or bacteria can promote antibody (Ab) isotype switching to IgG2a/c. Multiple cell types are capable of responding to TLR stimulation in vivo and the processes underlying TLR-induced Ab isotype switching are not fully defined. Here, we used feeble mice, which are deficient in the peptide/histidine transporter solute carrier family 15 member 4 (Slc15a4), and fail to produce cytokines including interferon alpha (IFNα) in response to TLR9 stimulation, to study Ab isotype switching to IgG2c in response to vaccination. We demonstrate that the production of IgG2c in response to CpGA-adjuvanted vaccines was severely reduced in feeble mice, while a more subtle defect was observed for CpGB. The reduced IgG2c production in feeble could not be ascribed to defective plasmacytoid dendritic cell (pDC) responses alone as we found that splenic cDCs and B cells from feeble mice were also defective in response to TLR9 ligation ex vivo. We conclude that Slc15a4 is required for intact function of TLR9-expressing cells and for effective Ab isotype switching to IgG2c in response to CpG-adjuvanted vaccines.

Independent expansion of epitope-specific plasma cell responses upon HIV-1 envelope glycoprotein immunization.

Abs that bind the functional envelope glycoprotein (Env) spike are considered critical for a broadly effective prophylactic HIV-1 vaccine. The difficulty in eliciting such Abs by vaccination is partially attributed to the immunodominance of hydrophilic, surface-exposed variable protein regions of Env. However, little is known about the potential for competition between B cells that recognize distinct and distal epitopes on Env during protein subunit vaccination. In this study, we address this basic question at the level of Ab-secreting cells and serum IgG using a pair of isogenic soluble Env trimers, designated wildtype and gV3, which differ only in their potential to activate B cell responses against the highly immunogenic V3 region of Env. Immunization of mice with gV3 resulted in a markedly lower Ag-specific response compared with that induced by wildtype Env and could be explained by a loss of V3-directed reactivities. There was no redistribution of the response to other regions of Env in gV3-inoculated mice, suggesting that the epitope-specific Ab-secreting cell responses measured after boost are independently regulated rather than dictated by direct or indirect competition between B cells recognizing different structural elements of Env. This information is relevant for ongoing efforts in Env immunogen design to focus responses on conserved neutralizing determinants and for our general understanding of B cell responses to large-protein Ags that display numerous B cell epitopes.

Human plasmacytoid dendritic cells efficiently capture HIV-1 envelope glycoproteins via CD4 for antigen presentation.

Advances in HIV-1 vaccine clinical trials and preclinical research indicate that the virus envelope glycoproteins (Env) are likely to be an essential component of a prophylactic vaccine. Efficient Ag uptake and presentation by dendritic cells (DCs) is important for strong CD4(+) Th cell responses and the development of effective humoral immune responses. In this study, we examined the capacity of distinct primary human DC subsets to internalize and present recombinant Env to CD4(+) T cells. Consistent with their specific receptor expression, skin DCs bound and internalized Env via C-type lectin receptors, whereas blood DC subsets, including CD1c(+) myeloid DCs, CD123(+) plasmacytoid DCs (PDCs), and CD141(+) DCs exhibited a restricted repertoire of C-type lectin receptors and relied on CD4 for uptake of Env. Despite a generally poor capacity for Ag uptake compared with myeloid DCs, the high expression of CD4 on PDCs allowed them to bind and internalize Env very efficiently. CD4-mediated uptake delivered Env to EEA1(+) endosomes that progressed to Lamp1(+) and MHC class II(+) lysosomes where internalized Env was degraded rapidly. Finally, all three blood DC subsets were able to internalize an Env-CMV pp65 fusion protein via CD4 and stimulate pp65-specific CD4(+) T cells. Thus, in the in vitro systems described in this paper, CD4-mediated uptake of Env is a functional pathway leading to Ag presentation, and this may therefore be a mechanism used by blood DCs, including PDCs, for generating immune responses to Env-based vaccines.

BLyS-mediated modulation of naive B cell subsets impacts HIV Env-induced antibody responses.

Neutralizing Abs provide the protective effect of the majority of existing human vaccines. For a prophylactic vaccine against HIV-1, broadly neutralizing Abs targeting conserved epitopes of the viral envelope glycoproteins (Env) are likely required, because the pool of circulating HIV-1 variants is extremely diverse. The failure to efficiently induce broadly neutralizing Abs by vaccination may be due to the use of suboptimal immunogens or immunization regimens, or it may indicate that B cells specific for broadly neutralizing Env determinants are selected against during peripheral checkpoints, either before or after Ag encounter. To investigate whether perturbation of B cell subsets prior to immunization with recombinant Env protein affects the vaccine-induced Ab response in mice, we used B lymphocyte stimulator (BLyS), a cytokine that regulates survival and selection of peripheral B cells. We show that the transient BLyS treatment used in this study substantially affected naive B cell populations; in particular, it resulted in more B cells surviving counter-selection at the transitional stages. We also observed more mature naive B cells, especially marginal zone B cells, in BLyS-treated mice. Intriguingly, provision of excess BLyS prior to immunization led to a consistent improvement in the frequency and potency of HIV-1 Env vaccine-induced neutralizing Ab responses, without increasing the number of Env-specific Ab-secreting cells or the Ab-binding titers measured after boosting. The results presented in this article suggest that an increased understanding of BLyS-regulated processes may help the design of vaccine regimens aimed at eliciting improved neutralizing Ab responses against HIV-1.

Immunization of macaques with soluble HIV type 1 and influenza virus envelope glycoproteins results in a similarly rapid contraction of peripheral B-cell responses after boosting.

The envelope glycoproteins (Env) represent a critical component of a successful antibody-mediated human immunodeficiency virus type 1 (HIV-1) vaccine. However, immunization with soluble Env was reported to induce short-lived antibody responses, suggesting that Env has unusual immunogenic properties. Here, we directly compared the magnitude and durability of B-cell responses induced by HIV-1 Env and an unrelated soluble viral protein, influenza virus hemagglutinin (HA), in simultaneously inoculated macaques. We demonstrate robust peak responses followed by rapid contraction of circulating antibody and memory B cells for both antigens, suggesting that short-lived responses are not unique to HIV-1 Env but may be a common feature of soluble protein vaccines.

Selective expansion of HIV-1 envelope glycoprotein-specific B cell subsets recognizing distinct structural elements following immunization.

The HIV-1 envelope glycoprotein (Env) functional spike has evolved multiple immune evasion strategies, and only a few broadly neutralizing determinants on the assembled spike are accessible to Abs. Serological studies, based upon Ab binding and neutralization activity in vitro, suggest that vaccination with current Env-based immunogens predominantly elicits Abs that bind nonneutralizing or strain-restricted neutralizing epitopes. However, the fractional specificities of the polyclonal mixture of Abs present in serum, especially those directed to conformational Env epitopes, are often difficult to determine. Furthermore, serological analyses do not provide information regarding how repeated Ag inoculation impacts the expansion and maintenance of Env-specific B cell subpopulations. Therefore, we developed a highly sensitive Env-specific B cell ELISPOT system, which allows the enumeration of Ab-secreting cells (ASC) from diverse anatomical compartments directed against different structural determinants of Env. In this study, we use this system to examine the evolution of B cell responses in mice immunized with engineered Env trimers in adjuvant. We demonstrate that the relative proportion of ASC specific for defined structural elements of Env is altered significantly by homologous booster immunizations. This results in the selective expansion of ASC directed against the variable regions of Env. We suggest that the B cell specificity and compartment analysis described in this study are important complements to serological mapping studies for the examination of B cell responses against subspecificities of a variety of immunogens.

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.

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 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.