High-Resolution Definition of Vaccine-Elicited B Cell Responses Against the HIV Primary Receptor Binding Site
ABSTRACT The high overall genetic homology between humans and rhesus macaques, coupled with the phenotypic conservation of lymphocyte populations, highlights the potential use of nonhuman primates (NHPs) for the preclinical evaluation of vaccine candidates. For HIV-1, experimental models are needed to identify vaccine regimens capable of eliciting desired immune responses, such as broadly neutralizing antibodies (bNAbs). One important neutralization target on the HIV-1 envelope glycoproteins (Envs) is the conserved primary CD4 receptor binding site (CD4bs). The isolation and characterization of CD4bs-specific neutralizing monoclonal Abs (mAbs) from HIV-1-infected individuals have provided insights into how broadly reactive Abs target this conserved epitope. In contrast, and for reasons that are not understood, current Env immunogens elicit CD4bs-directed Abs with limited neutralization breadth. To facilitate the use of the NHP model to address this and other questions relevant to human humoral immunity, we defined features of the rhesus macaque immunoglobulin (Ig) loci and compared these to the human Ig loci. We then studied Env-immunized rhesus macaques, identified single B cells expressing CD4bs-specific Abs, and sequenced and expressed a panel of functional mAbs. Comparison of vaccine-elicited mAbs with HIV-1 infection-induced mAbs revealed differences in the degree of somatic hypermutation of the Abs as well as in the fine specificities targeted within the CD4bs. These data support the use of the preclinical NHP model to characterize vaccine-induced B cell responses at high resolution.
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ABSTRACT: Studies in nonhuman primates offer information of high relevance to clinical medicine due to their close genetic relationship with humans. Here, we established an optimized protocol for the isolation of antibody V(D)J sequences from rhesus macaque B cells. Nested PCR primers were designed to align to sequences flanking the V(D)J coding region to enable amplification of highly mutated antibody sequences. The primers were evaluated using cDNA from bulk PBMCs as well as from single-sorted memory and naïve B cells from several macaques to ascertain effective germline coverage. The nested PCR efficiency reached 60.6% positive wells for heavy chain amplification, 39.2% for kappa chain, and 23.7% for lambda chain sequences. Matching heavy and light chain sequences, indicating antibodies that potentially can be cloned, were obtained in 50% of the positive wells. Using these primers, we found that the efficiency and specificity of V(D)J amplifications were markedly improved compared to when primers designed for human Ab isolation were used. In particular, the amplification of recombined light chain VJ sequences was improved. Thus, we describe the design and testing of a new set of rhesus-specific primers that enable efficient and specific amplification of heavy, kappa and lambda V(D)J genes from single sorted B cells. The use of these primers will facilitate future efforts to clone and express rhesus macaque MAbs for genetic, functional and structural analyses.Journal of immunological methods 09/2012; 386(1-2):85-93. DOI:10.1016/j.jim.2012.09.003 · 2.01 Impact Factor
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ABSTRACT: Vaccine-induced protection is generally mediated by long-lived antigen-specific B cell responses. Most licensed vaccines target pathogens that display relatively low variability, but for highly variable pathogens, such as HIV-1, vaccine development is more challenging. This thesis is focused on understanding vaccine-induced B cell responses against the HIV-1 envelope glycoproteins (Env), a critical vaccine target. Information about the immunogenic properties of candidate Env immunogens remains limited and so far the elicitation of broadly neutralizing antibodies (bNAbs) were not reported for any vaccine regimen tested in primates. Thus, there is a need to investigate vaccine-induced B cell responses against Env in more detail and to identify means to improve upon current Env-based vaccine strategies. Here, I investigate B cell responses in nonhuman primates immunized with soluble HIV-1 Env trimers to address these questions, as well as to gain an enhanced understanding about B cell responses to complex protein antigens in general. In paper I we established several assays for the evaluation of B cell responses in macaques. Following immunization with soluble trimeric Env, we comprehensively analyzed the B cell responses in the periphery, bone marrow, and mucosal compartments and further evaluated the elicited Abs for neutralization activity and protection in a SHIV challenge model. We observed high levels of Env-specific B cell responses following immunizations, improved breadth of neutralization compared to responses elicited by a monomeric Env vaccine tested in humans and delayed acquisition of SHIV infection compared to in control immunized animals. In paper II we evaluated longitudinal B cell responses following immunization with soluble trimeric Env and influenza HA protein, the latter included for comparative purposes. We found that peripheral B cell responses declined rapidly following boost, while antigen-specific long-lived plasma cells were stable for >6 months following immunization, for both antigens. In paper III we established a system for high-resolution evaluation of B cell responses in nonhuman primates. We first characterized the rhesus immunoglobulin loci to allow analyses of Ab gene usage and somatic hypermutation. We next isolated monoclonal antibodies (MAbs) targeting the HIV-1 primary receptor binding site (CD4bs) on Env and we examined the binding specificities of these Abs compared to infection-induced MAbs to unravel limitations of current vaccine-induced responses. In paper IV we optimized the RT-PCR method used in paper III for isolation of Ab V(D)J sequences from rhesus macaque B cells to facilitate future use of the macaque model for B cell studies. In conclusion, this thesis establishes several methods for the evaluation of B cell responses in nonhuman primates and it demonstrates that the soluble HIV-1 Env trimers induce potent, but relatively short-lived peripheral B cell responses. Additionally, we describe, for the first time, a set of vaccine-induced CD4bs-directed MAbs and we characterize their binding and neutralizing properties and discuss the implications of these results for improved Env vaccine design.11/2012, Degree: PhD in Medical Sciences, Supervisor: Gunilla Karlsson Hedestasm
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ABSTRACT: The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.PLoS ONE 06/2013; 8(6):e66276. DOI:10.1371/journal.pone.0066276 · 3.53 Impact Factor