Browsing by Subject "HIV vaccine"

This thesis focuses on modeling the selection effects on B cell antibody mutations to identify amino acids under strong selection. Site-wise selection coefficients are parameterized by the fitnesses of amino acids. First, we conduct simulation studies to evaluate the accuracy of the Monte Carlo p-value approach for identifying selection for specific amino acid/location combinations. Then, we adopt Bayesian methods to infer location-specific fitness parameters for each amino acid. In particular, we propose the use of a spike-and-slab prior and implement Markov chain Monte Carlo (MCMC) algorithms for posterior sampling. Further simulation studies are conducted to evaluate the performance of the proposed Bayesian methods in inferring fitness parameters and identifying strong selection. The results demonstrate the reliable inference and detection performance of the proposed Bayesian methods. Finally, an example using real antibody sequences is provided. This work can help identify important early mutations in B cell antibodies, which is crucial for developing an effective HIV vaccine.

Since its outbreak in the 1980s, the Human Immunodeficiency Virus (HIV) has become a major threat to global health. According to UNAIDS 2021 epidemiological estimates (2, 3), at the end of 2020 around 37.7 million people were living with HIV worldwide and 1.5 million new HIV infections occurred. Among those HIV-positive individuals, 10.5 million including 800,000 children did not have access to treatment, resulting in 680,000 AIDS-related deaths. Currently, different preventative measures are implemented including condom use, and pre- and post-exposure prophylaxis (4). However, these measures are not feasible for scaling up to population level because they require constant use of medication, and their efficacy depends on adherence to the treatment regimen. Vaccines are among the most cost-effective approaches to prevent infectious diseases (5). Yet, after 40 years of pandemic HIV, no vaccine has been able to provide enough protection against HIV acquisition, reflecting the formidable challenge of developing an efficacious HIV vaccine. These difficulties come from the two sides of the battle: the virus and the immune system. Virologically, HIV represents one of the most genetically diverse viruses in human history (6). Therefore, an effective vaccine will need to provide protection against an enormous set of viral strains. Moreover, the extensive glycosylation of the HIV Envelop glycoproteins (Env) protects the sites of vulnerability in the Env against antibody recognition (7), resulting in delayed emergence of neutralizing antibodies following infection (8, 9). Immunologically, antibodies with extensive neutralizing ability (known as broadly neutralizing antibodies, bnAbs) have features that disfavor their generation, such as high frequency of somatic hypermutation (SHM) (10), long complementarity determining region 3 (CDR3) (11), and high frequency of immunologically disfavored mutations (known as improbable mutations) (1). Because traditional vaccination strategies have failed to elicit broad protective immune responses, novel approaches are required for a universal HIV vaccine. The success of passive immunization with bnAbs in protecting non-human primates from the Chimeric Simian-human Immunodeficiency virus (SHIV) infection (12-17), has highlighted bnAbs as critical for protection; and elicitation of bnAbs is the ultimate goal for most current HIV vaccine designs. The features of bnAbs indicate that several rounds of SHM are required for B cells to produce bnAbs, and it is thought that the first event to drive B cells toward bnAb formation is when Env engages the B cell precursors that express the unmutated form of a bnAb, known as germline or unmutated common ancestor (UCA) (10). Different strategies have been used to engage the germline B cells such as using Env antigen of the transmitted/founder (T/F) virus that first establishes infection (18). Moreover, the use of native-like Env trimers instead of monomeric glycoproteins may enhance the immunogenicity of epitopes important for broad neutralization. To induce neutralizing antibodies against HIV, one of the vaccine candidates we tested (CH505T/F SOSIP) is a native-like trimer Env antigen designed based on the T/F virus isolated in a patient who developed the anti-CD4 binding site (CD4bs) bnAb CH103 (18). This antigen was chosen because it has demonstrated the ability to engage the CH103 UCA (19). While the mechanism of elicitation of bnAbs during natural infection are not completely elucidated, several recent studies have reported that HIV infected children develop neutralization breadth earlier and more frequently than adults (20, 21). Moreover, the few pediatric bnAbs described in the literature feature lower SHM frequency and fewer improbable mutations than adult bnAbs with similar neutralizing breath (1, 22, 23). Overall, these observations suggest that immunization in early life could present advantages for inducing bnAbs. Therefore, in one of the studies presented in this dissertation, we tested our immunization strategy in infant non-human primates. To date, only one HIV vaccine trial has demonstrated any evidence of efficacy. In the HIV vaccine trial RV144, the vaccine was correlated with a 31% reduction of HIV acquisition in the vaccinees (24). Importantly, only very limited neutralizing antibody response was induced in RV144 vaccinees, suggesting that non-neutralizing antibody functions can provide some degree of protection. Indeed, post-hoc analyses indicate that antibody-dependent cellular cytotoxicity (ADCC) was associated with reduced HIV acquisition in RV144 vaccinees (24, 25). Unlike neutralization, which is mainly regulated by the Fab region of an antibody, non-neutralizing antibody effector functions (such as ADCC and antibody-dependent cellular phagocytosis, ADCP) are regulated both by Fab and Fc regions (26, 27). Antibodies with a certain Fc phenotype exert non-neutralizing functions via selectively engaging different Fc receptors. (26, 27). As an ideal vaccine would probably need to induce antibodies that mediate neutralization as well as non-neutralizing Fc effector functions, it is important to further explore how vaccine strategies can modulate the Fc portion of the antibodies in order to enhance desired Fc functions. As traditional immunization strategies have failed to induce protective immunity against HIV, various classes of nanomaterials have been applied to HIV vaccine development in the past decade (28). That is because certain properties of nanomaterials can be harnessed to improve specific immune responses. Notably, self-assembling peptide nanomaterial such as the synthetic peptide Q11 which can self-assemble into fibrillar structure upon transition from pure water to aqueous solution with physiological salt concentration (29) are self-adjuvanting (30). Moreover, Q11 allows control of valency of the conjugated antigens (31). Thus far, Q11 has been used in immune therapy for different disease models such as malaria (32), influenza virus (33-35), psoriasis (36), and bacterial endotoxin-induced anaphylactic shock (37). Because of Q11’s engineerability and self-adjuvanting nature, it may present advantages for tailoring immune response against diseases with unknown immune correlates for protection such as HIV. The overarching goal of this dissertation project is to construct a next-generation HIV vaccine capable of inducing a broad protection against different HIV strains. We hypothesized that presenting HIV Env on Q11 improves the humoral response elicited by the Env vaccine. To test this hypothesis, we utilized Q11 to formulate different HIV vaccines, and we defined the humoral responses induced by Q11-based HIV vaccines in animal models including mice, rabbits, and infant rhesus macaques. We first conjugated gp120 from the T/F clade C virus 1086.C (38) to Q11 nanofiber and immunized wildtype mice with either the Q11-conjugated gp120 (gp120-Q11) or with soluble gp120. We demonstrated that gp120-Q11 induced higher magnitude of antibody response than gp120. More importantly, by testing the antibody binding to Envs from heterologous HIV strains, we demonstrated that gp120-Q11 also induced higher binding breadth. This enhancement in antibody binding magnitude and breadth was found to be associated with the gp120 valency on Q11 nanofiber, as diminished response was observed in mice immunized with lower valency gp120-Q11 vaccine. We then immunized rabbits with a gp120-Q11 and the corresponding gp120 to assess the function of the vaccine-elicited antibodies. The gp120-Q11 vaccine induced higher levels of tier 1 autologous virus (CH505 w4.3) neutralization, ADCC, and ADCP to heterologous Env antigen in rabbits than the gp120 vaccine. Since Fc-mediated functions such as ADCC and ADCP can be modulated by the glycosylation of the IgG Fc region, we analyzed the Fc glycan in gp120-specific IgG in the immunized rabbits and found that the gp120-Q11 vaccine induced an IgG response with higher levels of fucosylation, bisection, and mono-galactosylation. Similar glycosylation profile was also observed in gp120-Q11 immunized mice. These results suggest that Q11 nanofiber’s ability to modulate Fc glycosylation may be applicable across different mammalian species. Finally, we assess the immunogenicity of a Q11-conjugated trimeric Env construct (SOSIP) in infant rhesus macaques. Although slightly lower antibody magnitude was induced by the Q11-conjugated SOSIP vaccine as compared to SOSIP only, we found higher titers of neutralizing antibody against the autologous tier 1 HIV CH505 w4.3 in infant macaques immunized with the SOSIP-Q11 vaccine. Yet, SOSIP-Q11 and soluble SOSIP vaccine groups demonstrated similar frequency of neutralization of the autologous tier 2 virus (CH505T/F). These results may reflect differences in the animal models or on how neonatal and adult B cells respond to multivalent vaccines. Future studies should investigate the mechanism of this difference in order to define if construct optimization can improve the response to HIV Env-Q11 multivalent vaccines in pediatric settings. Taken together, in this dissertation we described a self-assembling nanomaterial as a versatile vaccine platform for HIV. This vaccine platform did not only improve the overall binding antibody response and breadth; antibodies induced by Q11 vaccine also demonstrated superior functional capacity (ADCC, ADCP and tier 1 virus neutralization). Moreover, Q11 appears to modulate the Fc glycosylation across species and these changes in glycosylation profile were associated with increased ADCC in rabbits. This finding implies an opportunity to further explore the possibility of using Q11 or other similar nanomaterials to tailor Fc-mediated antibody functions via modulating the glycan moiety.

The HIV-1 envelope (Env) glycoprotein trimer expressed on the virion surface is the target for both non-neutralizing and neutralizing antibodies (nAbs). In both infection and vaccination, the dominant neutralizing antibody responses are strain-specific, however, during chronic infection, antibodies that can potently neutralize genetically diverse HIV-1 isolates have been identified. These antibodies have been termed broadly neutralizing antibodies (bnAbs). BnAbs target conserved sites on the HIV-1 Env glycoprotein trimer including the membrane proximal external region, the gp120-gp41 interface, the CD4 binding site, V1V2-loop plus glycans, and the V3 loop plus glycans. Passive infusion studies with broadly neutralizing antibodies (bnAbs) of various specificities have been shown to be protective as well as control viral load.

Thus, one component of a protective HIV-1 vaccine will likely require the induction of broad and potent neutralizing antibodies, however, it is not understood how such a response would be elicited through vaccination as these antibodies are rare and restricted by immune tolerance mechanisms due to their unusual features such as extensive somatic hypermutation, presence of a long HCDR3, glycan-reactivity, and poly- and or auto-reactivity. Due to the counter selection of B cells bearing antibodies with these characteristics by the host immune system, bnAb precursor B cells are rare in the B cell repertoire and would likely be difficult to engage through vaccination. As such, vaccinations using HIV-1 Env have induced dominant strain-specific antibody responses, but not broadly neutralizing antibody responses. To efficiently engage and expand bnAb-precursor B cells, production of stable homogeneous immunogens that selectively express bnAb epitopes may be necessary.

Following the Introduction (Chapter 1), Methods (Chapter 2), Chapter 3 of this dissertation describes antibody reagents that are used to characterize recombinantly-produced HIV-1 Env glycoproteins. Chapters 4 and 5 present data relating to the ontogeny of vaccine-induced antibody responses to the first and second variable loops of the Env glycoprotein plus glycan (V1V2-glycan epitope) while Chapters 6 and 7 present data relating to the ontogeny of vaccine-induced antibody responses to the third variable loop plus glycan (V3-glycan epitope).

Chapter 4 of this dissertation describes a study of the antibody responses in rhesus macaques following immunization with a synthetic glycopeptide mimic of the epitope bound by antibodies that target the first and second variable loops plus glycan (V1V2-glycan bnAb epitope). This V1V2 glycopeptide induced robust plasma antibody binding responses to HIV Env that contained the same V1V2 loop sequence as the immunogen and binding of plasma antibodies to HIV Env was dependent on a lysine at position 169 (K169). Dependency on K169 is common for antibodies that target the V1V2 loop, such as the strain-specific antibodies CH58 and CH59, that recognize only a peptidic epitope of the V1V2 loop, but also for V1V2-glycan bnAbs such as CH01 that recognize both the V1V2 loop peptide backbone in addition to glycans at positions N156 and N160). Even though binding of vaccine-induced antibodies was dependent on the presence of K169, no glycan-reactive responses were detected, thus suggesting the presence of strain-specific, peptide-reactive CH58 and CH59-like antibodies.

Moreover, the heavy and light chain sequences of vaccine-induced antibodies were isolated and majority of Env-reactive antibodies paired with the rhesus Vλ3-17 lambda chain gene segment. It has been previously demonstrated that the rhesus Vλ3-17 lambda chain gene segment is the rhesus ortholog to the human Vλ3-10 gene as used by the strain-specific vaccine-induced human antibody, CH59. This is due to a germline-encoded glutamic acid and aspartic acid motif in the antibody HCDR2 loop (ED motif). The ED motif forms a salt bridge with the HIV-1 Env K169 and thus generates germline-encoded HIV-1 Env reactivity. In summary, immunization with the V1V2 glycopeptide minimal immunogen that was designed to target and expand rare V1V2-glycan reactive precursors elicited a dominant antibody response that solely recognized peptide, but not glycan. Thus, these results demonstrate that despite vaccinating with the V1V2-glycopeptide that preferentially expressed the V1V2-glycan epitope, V1V2 antibody responses that recognized only peptide were profoundly immunodominant. As V1V2-glycan bnAbs typically have long HCDR3 loops and long HCDR3-bearing B cells are rare in the peripheral B cell repertoire, the observed results could be due to lack of B cells that recognize the V1V2-glycan epitope. From this work, however, it is unclear if the observed results were due to a deficiency of these types of B cells in the periphery or due to instability of the vaccine immunogen in vivo.

Chapter 5 describes the characterization and immunization of a humanized mouse model bearing the CH01 V1V2-glycan bnAb unmutated common ancestor antibody; i.e. the B cells in these mice bear the CH01 antibody heavy and light chain sequences that were predicted to be germline-encoded prior to somatic hypermutation (germline-reverted). With a long HCDR3 of 24 amino acids, B cells bearing the germline-reverted CH01 heavy and light chain were not deleted in the bone marrow, and peripheral B cell development was comparable to the background strain, C57BL/6, demonstrating that B cells bearing CH01-like antibodies could be responsive to an appropriately designed vaccine immunogen.

Mice bearing only the germline reverted CH01 heavy chain were then immunized with HIV-1 Env. Immunization of these mice expanded B cell populations that were dependent on the presence of the V1V2-loop glycan N160. Furthermore, immunization of these mice elicited neutralization against difficult to neutralize (tier 2) HIV-1 isolates. Following immunization, B cells bearing antibodies whose binding to HIV-1 Env was N160 glycan-dependent were isolated, and heavy and light chain sequences were recovered. Analysis of the recovered heavy and light chains revealed that 18 different murine variable-kappa chain genes paired with the knocked in CH01 UCA and these pairings conferred N160 dependence. However, the knocked CH01 UCA heavy chain sequences were largely unmutated. These data suggest that long HCDR3-bearing B cells that are specific for the V1V2-glycan epitope (dependent on the presence of glycan at N160) are not deleted in the bone marrow and as such, vaccine-elicitation of antibodies targeting the V1V2-glycan epitope in wild-type animals should be feasible. Moreover, these data suggest that a critical first step for the elicitation of V1V2-glycan targeting antibody responses will be expansion of the B cells from which these responses derive, but additional antigenic diversity will likely be required to induce the full neutralization breadth and potency observed by V1V2-glycan bnAbs isolated from HIV-1 infected donors.

Chapter 6 details the characterization of a stable synthetic glycopeptide that was designed to mimic the conformation of the peptide component of the HIV-1 V3-loop plus glycans, termed “Man9-V3.” Broadly neutralizing antibodies that recognize this epitope, termed “V3-glycan bnAbs” bound to Man9-V3 glycopeptide and bound with affinities comparable to those observed for native-like gp140 Env trimers. Moreover, both fluorophore-labeled Man9-V3 and native-like trimers similarly bound to bnAb memory B cells, and by flow sorting, members of a V3-glycan bnAb clonal lineage from an HIV-1-infected individual were isolated. Thus, these data suggest that Man9-V3 glycopeptide is a structural mimic of the HIV-1 Env epitope bound by V3-glycan bnAbs and is a candidate immunogen to initiate V3-glycan bnAb lineage maturation.

In Chapter 7, the immunogenicity of Man9-V3 glycopeptide was tested in rhesus macaques. Using Man9-V3 as an immunogen, V3-glycan antibody responses were elicited. Combining flow sorting with next generation sequencing of immunoglobulin genes, the ontogeny of a vaccine-elicited V3-glycan antibody lineage, termed DH717 was studied. This lineage targeted the base of the HIV-1 V3-loop in addition to the V3-loop N301 and N332 glycans. Neutralization however, was limited to Env pseudoviruses bearing only high-mannose glycans as these antibodies could not neutralize viruses bearing native glycoforms. The structure of the most broad and potent member of the DH717 lineage, DH717.1 was determined using X-ray crystallography. This revealed that the rhesus DH717.1 V3-glycan antibody and 2G12, a human V3-glycan bnAb isolated from a HIV-1 infected donor, showed remarkable similarity in accommodation of high-mannose glycans. Specifically, DH717.1 and 2G12 antibodies accommodate terminal branches of high-mannose glycans through the formation of a binding pocket comprised of the HCDR1 and HCDR2 loops. Furthermore, DH717, like 2G12, bound the yeast Candida albicans in a glycan-dependent manner.

With regards to the ontogeny of the V3-glycan DH717 lineage, next generation sequencing at pre-immunization time points revealed the DH717 lineage to be present prior to vaccination and to be mutated with regards to the computationally inferred DH717 germline sequence. While the already mutated pre-vaccination DH717 lineage member bound to both the yeast Candida albicans, and to Man9-V3 glycopeptide, the DH717 unmutated common ancestor bearing a computationally inferred germline sequence bound only to Candida albicans, and not Man9-V3. It was only after acquiring somatic mutations prior to immunization did the lineage acquire the ability to bind to Man9-V3, suggesting a role for high-mannose-bearing environmental antigens for priming such responses. After further acquisition of somatic mutations following vaccination did the lineage acquire the ability to bind the V3-glycan bnAb epitope as presented on a stabilized, native-like soluble recombinant HIV-1 Env trimer. This suggests that vaccination with a synthetic glycopeptide affinity matured a pre-existing, yeast-reactive B cell lineage to the HIV-1 V3-glycan bnAb epitope.

Together, the studies described in Chapters 4-7 suggest that vaccine-induction of V1V2- and V3-glycan bnAbs may be feasible, and that to do so, stable glycopeptides that mimic bnAb epitopes will be needed to select for and expand the precursors for the desired glycan-bnAb response.