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- Sundling, Christopher
(författare)
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Dissection of HIV-1 Env-specific B cell responses in non-human primates
- 2010
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- It has been almost 30 years since the identification of HIV as the causative agent of AIDS. Despite considerable efforts to halt the spread of the virus, the epidemic continues in many parts of the world. The most efficient way to stop new infections is a prophylactic vaccine that blocks transmission at the viral portal of entry. To generate an efficacious vaccine, neutralizing antibodies directed against the envelope glycoproteins (Env), the only virally expressed proteins on the surface of the virion, likely need to be elicited. Early attempts to develop an HIV vaccine used soluble recombinant monomeric Env. This vaccine candidate generated high titers of Env binding antibodies in a large clinical trial; however, no protection against infection was observed. Since then attempts have been made to design Env immunogens that more closely mimic the native Env spike with the aim to improve the quality of vaccine-elicited humoral immune responses. Due to the lack of high resolution three dimensional structures of Env in its native conformation, these design efforts are still empirical. To guide future immunogen design efforts, it is important to understand what current Env vaccine candidates elicit in relevant pre-clinical models, both at the serological and cellular level. The focus of this thesis has been to investigate B cell responses in non-human primates following immunization with soluble HIV-1 Env trimers. We show that Env immunization in adjuvant elicits high levels of binding antibodies as well as robust peripheral memory B cell and plasma cell populations. The kinetics and magnitude of the responses at early time points after immunization are similar to those elicited by the successful human influenza and rabies vaccines. Despite these potent responses, we only observed a modest protective effect upon mucosal SHIV challenge of vaccinated animals. When quantifying the levels of Env-specific antibodies in the mucosa we found them to be 1000-10 000 fold lower than in serum, which may explain the lack of protection in this heterologous challenge model. From in vitro studies it has been proposed that interactions between Env and CD4-expressing T cells may detrimentally affect T cell function. To investigate if in vivo interactions between Env immunogens and CD4-expressing cells would negatively affect the elicitation of Envspecific immune responses, CD4 binding-competent and CD4 binding-defective trimers were constructed and inoculated into non-human primates. No difference was observed between the groups in regards to Env-specific antibody titers, memory B cell or T cell levels and functionality, indicating that Env binding to CD4 in vivo was not detrimental to vaccine elicited responses. Furthermore, using the CD4 binding-competent and CD4 binding-defective Env immunogens, we assessed if in vivo CD4 binding affected the outcome of an intravenous SHIV challenge. Antibodies directed against the conserved co-receptor binding site of Env were only observed following immunization with the CD4-binding competent trimers. However, the presence or absence of this class of antibodies did not influence the level of protection against SHIV infection. These results indicate that Env-CD4 in vivo interactions, and specifically co-receptor binding site-directed antibodies, did not contribute to the control of viremia observed in this challenge model.
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| 2. |
- Sundling, Christopher
(författare)
-
Dissection of HIV-1 Env-specific B cell responses in nonhuman primates
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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 highresolution 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.
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