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Over three decades have passed since the discovery of HIV-1, yet an AIDS vaccine remains elusive. The envelope glycoprotein of HIV-1 gp120, is the most exposed protein on the viral surface and thus serves as an important target for vaccine design. However, various factors like high mutability of gp120, extensive glycosylation and very high conformational flexibility of gp120 have confounded all efforts to design a suitable immunogen that elicits broad and potent neutralizing antibodies against HIV-1. In Chapter 1, a brief description of the structural organization of HIV-1 along with the progress made and the difficulties encountered in the development of a vaccine are presented. In Chapter 2, the design and characterization of an outer domain immunogen of HIV-1 gp120 is discussed. The outer domain (OD) of the envelope glycoprotein gp120 is an important target for vaccine design since it contains a number of conserved epitopes, including a large fraction of the CD4 binding site. Attempts to design OD based immunogens in the past have met with little success. In this work, we designed an OD immunogen based on the sequence of the HXBc2 strain, expressed and purified it from E. coli (ODEC). The ODEC molecule lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gp120 to increase solubility. Biophysical studies showed that ODEC is folded and protease resistant while ODEC lacking the designed mutations is highly aggregation prone. In contrast to previously characterized OD constructs, ODEC bound CD4 and the broadly neutralizing antibody b12 with micromolar affinities, but not the non-neutralizing antibodies b6 and F105. Further improvement in the refolding protocol yielded a better structured molecule that bound CD4, b12 and VRC01 with sub-micromolar affinities. In rabbit immunization studies with animals primed with ODEC and boosted with gp120, the sera are able to neutralize Tier I viruses and some Tier II viruses like JRFL and RHPA with measurable IC50s. This is one of the first examples of a gp120 fragment based immunogen which was able to elicit sera that showed modest neutralization of some Tier II viruses. Subsequently amide hydrogen-deuterium exchange studies of ODEC showed that though the molecule is well-folded, it is labile to exchange. This might indicate why ODEC does not elicit high amounts of neutralizing antibodies. In Chapter 3, we report the design and characterization of two smaller fragments of gp120 (b121a and b122a) to target the epitope of the broadly neutralizing antibody b12. The region chosen comprised of a compact beta barrel in the lower part of the outer domain of gp120. Unlike ODEC, the fragments corresponding to these constructs were not contiguous stretches in gp120. Thus we used linkers to connect them. Further, nine designed mutations were introduced at exposed hydrophobic regions of the fragment to increase its solubility. The designed protein fragments were expressed in E. coli in order to prevent glycosylation and consequent epitope masking that might occur if expressed in an eukaryotic expression system. Biophysical studies showed that b121a/b122a are partially folded. Disulfide mapping studies showed that the expected disulfide bridges were formed. The designed immunogens could bind b12, but not the non-neutralizing antibody b6. Sera from rabbits primed with b121a/b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity against a 20 virus panel including Tier2 and 3 viruses such as PVO4, CAAN, CAP45 and ZM233. Sera from animals that received only gp120 showed substantially decreased breadth and potency. Serum depletion studies confirmed that neutralization was gp120 directed and that a substantial fraction of it was mediated by CD4 binding site (CD4bs) antibodies. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals, despite the restricted germline VH gene usage observed so far in broadly neutralizing CD4bs directed antibodies in humans. In Chapter 3, we also discuss design of a new construct b122d, which includes regions corresponding to b121a, but with linker connectivities similar to b122a. It was found to bind b12 with sub-micromolar affinity and also showed proteolytic resistance comparable to b121a. This indicated that though b121a showed better proteolytic resistance than b122a, it bound b12 poorly because one of the linkers might sterically occlude the b12 binding site. As the b12 binding site constructs based on the subtype B HXBc2 sequence elicited neutralizing antibodies, we chose to design similar constructs based on a subtype C sequence. The proteins (Cb122a and Cb122d) were purified from E. coli, characterized and found to bind b12 with micromolar affinity. The new constructs (b122d, Cb122a, Cb122d) will shortly be tested in animal immunizations. Disulfides are known to stabilize proteins by reducing the entropy of the unfolded state. In Chapter 4, we attempted to stabilize b122a by engineering disulfides. The disulfides are expected to rigidify the molecule and possibly improve its ability to elicit neutralizing antibodies. Some of the disulfides tested in b122a were predicted based on stereo-chemical criteria by the program MODIP (Modeling Disulfide Bridges in Proteins), while others were chosen at non-hydrogen bonded positions (NHB) on anti-parallel beta strands, based on earlier studies in the lab. Some of the disulfide mutants showed better binding to b12 and increased protection to enzymatic digestion. These disulfides were subsequently engineered into other b12 binding site constructs, namely b122d, Cb122a and Cb122d and these were biophysically characterized. Amongst the various disulfides that were tested in b122a, the one at 293-448 (according to HxBc2 numbering) was found to improve the binding to b12 by about ~16-fold. Not only did this disulfide improve the binding of b122a to b12, it also showed similar improvement in case of b122d and both the subtype C constructs tested. Moreover, since the position 293-448 is an exposed NHB position of an anti-parallel beta strand, spontaneous formation of the disulfide and the improved binding to b12 for all the proteins tested reinforces the fact that cysteines engineered at such positions leads to formation of a stabilizing disulfide. All the proteins containing the 293-448 disulfide will be used in future for rabbit immunization studies to examine if they elicit better neutralizing antibodies than the parent b122a molecule. As discussed in Chapter 2, ODEC showed a very fast rate of hydrogen exchange, indicating that it is flexible. As the 293-448 disulfide improved the binding of b12 binding site constructs, in Chapter 5, disulfides at exposed NHB positions were introduced in the context of ODEC. Previously engineered inter-domain disulfides have been shown to reduce the conformational flexibility of gp120. The disulfides in the lower beta barrel of the outer domain which harbors the CD4 binding site were found to be monomeric, oxidized and could bind neutralizing CD4bs antibodies better than the WT protein. On the other hand, the disulfides in the upper barrel of the outer domain were aggregated and bound antibodies poorly compared to the WT protein, indicating that this part of the molecule may not be well structured in the fragment. However, there was no significant change in the hydrogen exchange kinetics for these mutants. Mutations in the Phe-43 cavity of gp120 (S375W/T257S) which constrain gp120 in the CD4 bound conformation were also tested in ODEC (ODEC-CF). This protein was found to bind CD4 and VRC01 about 8 and 2 times better respectively than WT ODEC. These improved immunogens will be used shortly in rabbit immunization studies. In an attempt to improve the immunogenicity of the gp120 fragment proteins, b121a, b122a and ODEC were displayed on/conjugated to the surface of Qβ virus like particles in Chapter 6. Exposed single cysteine mutants of these proteins were purified, characterized biophysically and found to have the single cysteine free for conjugation. These were subsequently conjugated to the Qβ virus like particles through click chemistry (carried out in Prof. MG Finn’s lab at TSRI), purified and used for rabbit immunization studies. The gp120 ELISA titers of the elicited sera showed that conjugation may be a better option to display foreign antigens on the surface than genetic fusion. There was no difference in the ELISA titers with and without adjuvant, indicating that the particles are sufficiently immunogenic in themselves. Sera from these studies will be tested in neutralization assays. The overall utility of the particle based display approach will be assessed by comparing neutralization data from particle based immunizations to identical immunizations with unconjugated immunogens. Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the Env surface protein. Native Env consists of a trimer of gp120:gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs) directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. To understand this further, in Chapter 7, we have designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1/V2 loop. h-CMP-V1cyc is one such variant where 153 and 142 are the N and C terminal residues of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity relative to wtgp120. It binds the non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1 bound the V1V2 directed broadly neutralizing antibodies PG9/PG16 with ~20 fold higher affinity compared to wild type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support Env models in which the V1V2 loops are proximal to the trimer interface. In Appendix A1, peptide analogs of selected secondary structural elements of gp120 were designed. Some of them were grafted on known scaffold proteins. The synthesized peptides were characterized biophysically. Most of the peptides did not have a well-defined secondary structure, indicating that they are not stable in isolation. Hence they were not pursued for further studies. One helical peptide adopted a significant amount of structure in aqueous buffer and will be shortly conjugated to carrier proteins and used in immunization studies. In Appendix A2, we created error-prone PCR libraries and loop-randomization libraries of b12 binding site constructs and attempted to screen these for better b12 binding using phage-display. However the screening was unsuccessful as the phages showed non-specific binding to b12 antibody. These libraries will be screened in future using yeast display.

Influenza virus is a pleiomorphic human pathogen which causes self-limiting respiratory illness lasting one-two weeks in most individuals. However, in immunologically compromised individuals, influenza infection may lead to severe morbidity and fatality. Annual epidemics cause 250,000-500,000 deaths worldwide and remain a major public health threat. The virus has evolved mechanisms of antigenic ‘drift’ and ‘shift’ to evade the host immune response. Hence, current influenza vaccines need to be updated every few years. Moreover, the currently available inactivated/live attenuated vaccines entail virus culture in embryonated chicken eggs hindering rapid scale-up. The aforementioned limitations of the current vaccines has had debilitating effect when strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past, despite intensive monitoring. Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift, facilitating a potential pandemic outbreak. These concerns have expedited efforts towards developing a ‘universal’ flu vaccine. Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the mature, disulfide linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit while the stem domain predominantly comprises of the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH induced conformational rearrangement in the host-cell endosomes to adopt the virus-host membrane fusion competent state. The ‘antigenic sites’ on the immunodominant globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed neutralizing antibodies (nAbs). As opposed to the highly-variable head domain, the HA stem is conserved and targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes. Although several bnAbs bind to the conserved HA stem, focusing the immune response to this conserved, subdominant stem domain in presence of the variable head domain of HA has been challenging. Alternatively, mimicking the epitope of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogen capable of eliciting a broadly protective immune response has been difficult because of the metastable conformation of HA. Addressing the aforementioned challenges, we describe the design and characterization of novel influenza immunogens by HA protein minimization. Chapter 1 gives an overview of the influenza virus life cycle, and outlines the structural organization and function of viral proteins. The conventional vaccines that are currently used and their limitations are described in this chapter. Recent improvements in influenza vaccine production focusing on recombinant HA as an alternate solution are discussed. Painstaking efforts of several groups in the recent past has led to the isolation of bnAbs that recognize novel ‘antigenic signatures’ within the globular head and the HA stem domains. Attempts to focus the immune response to these ‘cross-protective’ epitopes are described. The design and characterization of trimeric HA stem-fragment immunogens from influenza A Group-1 viruses which mimic the native, pre-fusion conformation of HA are described in Chapter 2. We engineered ‘headless’ HA stem immunogens based on influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation sensitive stem-directed bnAbs such as CR6261, F10 and FI6v3 with high affinity (equilibrium dissociation constant [KD] of 10-50nM). The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both Group-1 (H1, H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD90) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens conferred robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous HA stem domain immunogens, the designed immunogens described here were purified from the soluble fraction in E.coli. These HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks. The detailed mechanism(s) by which our ‘headless’ stem immunogens provide protection need further investigation. The long central α-helices (LAH) located in the HA stem assemble together into a parallel, trimeric coiled-coil. Immunization with the wt-LAH (76-130 of HA2) derived synthetic peptide designed from an H3 subtype (H3N2 A/Hong Kong/1/68) and conjugated to keyhole limpet hemocyanin (KLH) was shown previously to elicit antibodies reactive in ELISA with multiple hemagglutinin subtypes and to confer protection against challenge with H3N2, H1N1 and H5N1 virus strains. The LAH peptide sequence was chosen based on maximal binding to the monoclonal antibody (MAb), 12D1, which has broad neutralizing activity against influenza viruses of the H3 subtype. These results motivated us to rationally design stabilized derivatives of wt-LAH and test their protective capacity in a mouse challenge model of influenza. This work is described in Chapter 3. Additionally, to understand the contribution towards protection conferred by the two distinct surface exposed patches on LAH, we designed constructs spanning different stretches of LAH. The biophysical characterization of the LAH-derived constructs indicates that most of them were well-folded. All these constructs were moderately immunogenic in mice but at best, conferred limited protection from lethal viral challenge. In contrast to previously reported results, our data suggests that the LAH in the absence of other regions of HA may require not only strong, but also specific adjuvantation to induce a robust and functional immune response in vivo. Chapter 4 describes an immunogen design (H1pHA9) based on the globular head domain of pandemic H1N1 HA which can be produced using a prokaryotic expression system. The HA-fragment, H1pHA9, stably refolds to mimic the conformation sensitive neutralizing epitopes in the globular head domain of HA. We have also successfully engineered the HA head domain to delineate the epitope of antibodies neutralizing the pandemic H1N1 virus using a yeast cell-surface display platform. In this direction, we report the isolation of a novel, neutralizing murine MAb, MA2077, against the pandemic H1N1 virus. The epitope of this MAb has been mapped onto the ‘Sa’ antigenic site. The ability of the head domain fragment, H1pHA9, which binds MA2077 with high affinity to elicit such neutralizing antibodies in vivo needs to be further explored. Structural analysis has shown that elements of the HA stem diverge between the two phylogenetic groups. Therefore, to mitigate the threat of circulating influenza A viruses from these distinct structural classes (H1 and H3 belonging to Groups 1 and 2 respectively), in lieu of a ‘universal’ vaccine, a combination of immunogens derived from both the groups is a practical alternative. In Chapter 5 we describe the design of stem-fragment immunogens from an influenza A Group-2 virus strain. We report the characterization of engineered ‘headless’ HA stem immunogens based on influenza A/Hong Kong/1/68 (H3N2) subtype. The designed immunogens were expressed in E.coli and purified from the soluble fraction with abundant yields (~15mg/lt). The HA stem-fragment immunogens could be concentrated to high concentrations without aggregation. While, H3HA10-IZ and H3HA10-Foldon, the trimeric derivatives of our parent construct (H3HA10) which were folded, conferred modest protection against a lethal homologous virus challenge in mice, there is considerable scope to improve our immunogen design. Analyzing the results from our previous work (Chapter 2), we speculate that structural elements at the N-terminus of A-helix are critical for helix initiation. We therefore extended the design to include residues from the start of the A-helix. We designed the extended stem immunogens from both H3 and H7 subtypes. The proteins were purified from the soluble fraction of the E.coli cell culture lysate. Preliminary studies suggest that extension of the A-helix has aided proper folding. These proteins need to be further characterized and evaluated in an animal model.

Influenza is an important respiratory pathogen that infects several million people each year. Currently available flu vaccines have to be updated regularly in order to be effective as the virus changes its composition by antigenic drift and shift. Most of the antibody response generated by these vaccines is strain specific as it is directed against the head domain (HA1) of HA. The HA2 subunit of hemagglutinin is highly conserved and immunogens designed from this subunit are likely to provide protection against multiple strains of the virus. However, expression of HA2 alone in the absence of HA1 resulted in a protein that took up the low pH conformation of HA. Our goal was to design immunogens from HA2 that would fold into the neutral pH form. Sequence analysis of a large number of HA protein sequences was carried out to identify conserved and exposed regions on HA. Several peptide and protein constructs were designed from the stem region of HA. These proteins were expressed in bacteria and purified proteins were used to immunize mice. Immunized mice were challenged with a lethal dose of virus to test for efficacy of the immunogen. Using this approach, stem domain constructs of HA were successfully designed and shown to take up the neutral pH form. These immunogens were also shown to be capable of providing broad range protection. Residues involved in the low pH induced conformational change of HA were identified from studies on HA2 derived peptides.

Influenza virus belongs to the Orthomyxovirus family of viruses that causes respiratory infection in humans, leading to morbidity and mortality. The mature influenza A virion has an envelope that contains two major surface glycoproteins proteins – hemagglutinin (HA) and neuraminidase (NA). HA is a highly antigenic molecules and is responsible binding to host cell surface receptors (Sialic acid), and membrane fusion between the viral membrane and the host endosomal membrane. Most of the antibody response generated against influenza virus either by vaccination or by natural infection is directed against HA. Influenza virus has segmented negative–sense RNA genome which gives the virus the ability to evade the host immune response by incorporating mutations (antigenic drift) and/or by reassotment with other subtypes of influenza A viruses (antigenic shift). Currently licensed vaccines which include an inactivated vaccine, a live attenuated vaccine, and recombinant subunit vaccine are beneficial for providing protection against seasonal influenza viruses that are closely related to the vaccine strain but fail to provide protection against drifted strains. This limits their breadth of protection and thus requires annual revaccination with reformulated vaccines. Also, because selection of a vaccine strain for the next season is purely based on surveillance and prediction, sometimes mismatches do happen between the selected vaccine strains and circulating viruses, resulting in a drastic decrease in vaccine efficacy and thus high morbidity and mortality. Furthermore, the production of these seasonal vaccines takes 6-8 months on an average, and does not guarantee protection against infection with novel reassortant viruses which can cause pandemics. To overcome the draw-backs of seasonal influenza virus vaccines and to enhance our pandemic preparedness, there is an increasing need for game-changing influenza virus vaccines that can confer robust, long-lasting protection against a broad spectrum of influenza virus isolates. Influenza hemagglutinin (HA) is highly immunogenic and thus a major target for vaccine design. HA is synthesized as a precursor polypeptide (HA0), assembles into a trimer, matures by proteolytic cleavage along the secretory pathway and is transported to the cell surface. Mature HA has a globular head domain, primarily composed of the HA1 subunit, which mediates receptor binding, while the stem domain, predominantly comprises of the HA2 subunit, and houses the fusion peptide. At neutral pH, the HA stem is trapped in a metastable state but undergoes an extensive conformational rearrangement at low pH in the late endosome (host-cell endosome) to trigger the fusion of virus and host membranes. Clusters of ‘antigenic sites’ have been identified in the head domain of HA, indicating that it harbors an almost continuous carpet of epitopes that are targeted by antibodies. However, these immunodominant sites constantly accumulate mutations to escape immune pressure, and thereby narrow the breadth of head-directed neutralizing antibodies (nAbs). In contrast to the highly-variable head domain, the membrane-proximal HA stem subdomain has much less sequence variability and, thus, is a desirable target for influenza vaccine development. In the recent past, several broadly neutralizing antibodies (bnAbs) targeting this subdomain with neutralizing activity against diverse influenza A virus subtypes have been isolated from infected people, further proving that this subdomain of HA can be targeted as a vaccine candidate. Steering the immune response towards this conserved, subimmunodominant stem subdomain in the presence of the variable immunodominant head domain of HA has been quite challenging. Alternatively, mimicking the epitome of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogenic capable of eliciting a broadly protective immune response has been difficult because of the metastable nature of HA. Addressing the aforementioned challenges, here we describe the design, stabilization and characterization of novel stem derived immunogens from HA of influenza A viruses using a protein minimization approach. Chapter 1 gives an overview of the influenza virus life cycle, nomenclature and classification of influenza virus; outlines the structural organization and functional properties of different viral proteins. An introduction to the kind of immune responses generated during vaccination or natural infection with the virus is discussed. The conventional vaccines that are currently used and their limitations, recent progress in the field of novel vaccine developmental approaches targeting the conserved epitopes on HA, is also described in this chapter. This chapter also gives a broad overview of bnAbs that have been isolated in the recent past, which target the novel antigenic signatures on HA. The design of a stem domain construct from an H3N2 virus (A/HK/68) is described in Chapter 2. In order to ensure that HA2 folds into the neutral pH conformation, regions of HA1 interacting with it were included in the design. Additionally, two Asp mutations were introduced in the B loop of HA2 to destabilize the low pH conformation and stabilize the desired native, neutral pH conformation. Studies using small peptides (57-98 of HA2) indicated that Asp mutations at positions 63 and 73 destabilized the low pH conformation. Studies on mutants with additional pairs of introduced Cys residues showed that the designed protein H3HA6 was folded into the neutral pH form. Immunization studies using mice showed that the protein was highly immunogenic and provided complete protection against a lethal dose of a homologous virus. Two constructs H3HA6a and H3HA6b, designed from the stem region of drifted H3N2 viruses (A/Phil/2/82 and A/Bris/10/07) were tested for protection against HK/68 to determine the extent of cross-strain protection provided by HA6. While HA6a (from A/Phil/2/82) provided near complete protection against HK/68, HA6b could protect against challenge only partially, possibly because of lower titers of antibodies elicited by this antigen. Studies using FcRγ chain knockout mice indicated that majority of the protection mediated by anti-HA6 antibodies was because of antibody mediated effectors functions, although neutralization as a mechanism of protection was also likely to contribute. In all the 18 subtypes of HA, the B loop contains residues that form the hydrophobic core of the extended coiled coil of the low pH form. As in the case of H3HA6, we suggest that these residues could be mutated to Asp to destabilize the low pH conformation. Two circularly permuted stem domain constructs from an H1N1 virus (A/PR/8/34) and an H5N1 virus (A/Viet/1203/04) were made. The design and characterization of these proteins is described in Chapter 3. H1HA6, H1HA0HA6 and H5HA6 were purified from inclusion bodies and refolded. The proteins H1HA6 and H1HA0HA6 were highly immunogenic and provided protection against a lethal challenge with homologous PR/8/34 virus. Anti-H1HA6 sera had higher titres of antibodies against heterogonous HAs as compared to convalescent sera. Stem derived immunogens from drifted H1N1 viruses (A/NC/20/99 and A/Cal/7/09) have been made and tested for cross-protection with PR/8/34 challenge. While H5HA6 also elicited high titers of antibodies, it could only protect partially against PR/8/34 challenge probably because high enough titers of cross-reactive protective antibodies were not elicited by this protein. These stem immunogens conferred robust subtype specific and modest heterosubtypic protection in vivo against lethal virus challenge. However, the immunogens, especially H1HA6, a stem immunogen from group 1 (PR8) virus is aggregation prone when expressed in E.coli. The strategy used to improve the biophysical and biochemical properties and thus the immunogenicity of these stem derived immunogens is discussed in Chapter 4. A random mutagenesis library of H1HA6 was constructed by error prone PCR using modified nucleotide analogues. The library was displayed on the yeast cell surface to isolate mutants showing better surface expression and improvement in binding to the broadly neutralizing antibody CR6261 compared to the wild-type protein. We isolated few clones, of which one mutant (H1HA6P2) dominated the enriched population. The other mutants differed slightly from H1HA6P2. This mutant differs from the wild-type by two mutations K314E and M317T (H1 numbering) which are close to the CR6261 binding site but outside the antibody foot-print (epitope). This mutant showed improved binding to CR6261 and exhibited significant improvement in surface expression. Improvement was also observed in binding of this mutant to F16v3-ScFv (another broadly neutralizing antibody). Two cysteine mutations were also introduced to further stabilize the trimeric form of the protein. Chapter 5 describes the biophysical and biochemical characterization of the high affinity isolated mutant at the protein level. We expressed this affinity matured mutant gene in E.coli and purified the protein from inclusion bodies. The stabilized mutant protein showed remarkable improvement in biophysical and biochemical properties and was recognized by stem directed conformation sensitive broadly neutralizing antibodies CR6261, F10 and F16v3 with affinity comparable to the full-length HA ectodomain. These results clearly suggest that this mutant protein is properly folded in its native pre-fusion conformation and thus can be an excellent candidate for eliciting stem directed broadly neutralizing antibodies. All these stabilized versions of stem derived immunogens will be tested for immunogenicity and cross-protection with different viral challenges. Chapter 6 describes the development of a method for mapping antibody epitopes (especially conformational epitopes) down to the residue level. Using a panel of single cysteine mutants, displayed on the yeast cell surface, this bypasses the need for laborious and time consuming protein purifications steps used in conventional methods for epitope mapping. We made a panel of single cysteine mutants, covering the entire surface of the antigen (CcdB, a bacterial toxin protein), displayed each mutant individually as well as in a pool, representing all mutants together on the yeast cell surface, and covalently labeled the cysteine with biotin-PEG2-maleimide to mask the area. The effect on antibody binding was monitored to identify the residues and relative positions important for antibody interactions with the displayed antigen by flow cytometry. By using this method we were able to map the conformational as well as linear epitopes of a panel of monoclonal antibodies down to the residue level with ease, and also identify the regions on the antigen which contribute to the antigen city during immunization in different animals. Since, this method is quite easy, rapid and gives in-depth information about antigenic epitopes, it can be useful in rational design of epitomes specific vaccines and other antibody therapeutics. It can easily be extended to other display systems and is a general approach to probe macromolecular interfaces.

The Human Immunodeficiency Virus (HIV) is a member of the retroviridae family from lentivirus genus which primarily infects CD4+ T cells and also to lesser degree monocytes, macrophages, and dendritic cells causing progressive failure of the immune system, ultimately leading to development of acquired immunodeficiency syndrome (AIDS). Currently ~ 37 million people are infected with HIV-1 with approximately 2 million new infections occurring every year (UNAIDS, 2016). Developing safe, effective, and affordable vaccines to prevent HIV infection is the best hope for controlling the HIV/AIDS pandemic. Envelope glycoprotein (Env) on the HIV-1 virion surface is synthesized as a single precursor protein gp160 which is cleaved by furin to form the gp120 and gp41 subunits. gp41 is inserted into the membrane, while gp120 remains non-covalently associated with the ectodomain of gp41 to form a trimer of heterodimers. gp120 binds to the CD4 receptor on CD4+ T cells, which triggers a series of conformational changes leading to the exposure of co-receptor binding sites on gp120. Subsequent binding to the co-receptor (CXCR4 or CCR5) on T-cells initiates fusion of cellular and viral membranes via gp41 subunit. The envelope glycoprotein gp120, on the virion surface is the most accessible component of HIV-1 to the host immune system, and the target of most of the neutralization response. However, the virus has evolved many efficient ways to escape this immune surveillance. Extensive glycosylation of gp120 is one way by which it masks critical neutralization epitopes and the presence of immunodominant long variable loops focuses the immune response away from conserved regions. Certain conserved epitopes are cryptic and get exposed only after gp120 binds to its receptor. Also gp120 and gp41 are highly flexible molecules, attached in a non-covalent fashion to form a trimer of heterodimers, leading to inherent metastability of the Env. This results in exposure of a large number of non-native conformations to the immune system and thus minimizes elicitation of neutralizing antibodies. Despite these defense mechanisms, about 20-30% of HIV-1 patients do generate a broad neutralization response. Although these bNAbs and their epitopes have been identified, eliciting similar bNAbs through immunization is challenging. Monomeric gp120 when used as an immunogen elicits non neutralizing antibodies. This indicates that the epitopes of bNAbs are not present in the right conformation on this molecule. A rational design approach which focuses the immune response towards specific epitopes targeted by bNAbs is required, with the aim to maximize the exposure of conserved neutralization epitopes and to simultaneously ensure minimal exposure of variable non neutralizing epitopes. This can likely be achieved either by (a) stabilization of native Env trimers, or/and by (b) protein fragment design. Chapter 1 gives a brief description of HIV-1 virus. Structural features of the Env protein are described along with epitopes targeted by various bNAbs. Various strategies employed towards structure based vaccine design are discussed. One of the strategies towards rational vaccine design is using protein fragment based approaches. Grafting epitopes onto heterologous scaffolds is a promising approach which can provide more structural stability to the epitope, helps focus immune response on the epitope of interest and can be employed in a prime boost strategy for immunization studies. In a scaffold based approach we used crystal structure information of gp120 in complex with bNAb b12 to define the epitope of this antibody. In Chapter 2 we use this epitope information to graft the epitope on an unrelated scaffold protein to design unique epitope scaffolds. We report a computational strategy to graft the discontinuous epitope of b12 antibody onto different scaffold proteins. Our strategy focuses on identifying the best match of the target scaffold to the query protein so as to cause the least structural disturbance in the scaffold protein. The best hits were screened for binding to b12 using Yeast Surface Display (YSD). Random mutant libraries were also generated to screen for better b12 binders using YSD. We further characterized a few of these epitope scaffolds after purifying them from bacterial systems. One of the epitope scaffolds 1mkh_E2 bound to b12 with a KD value of 7.5µM. 2bodx_03, an unoptimized epitope scaffold reported previously (Azoitei et al, 2011) binds b12 with a KD value of 300μM. Thus our epitope scaffold 1mkh_E2 shows reasonable binding to b12 without any optimization. We are currently purifying other b12 epitope scaffolds and will be characterizing them for binding to b12. We have previously used a protein minimization strategy to design fragments of gp120, called b122a and b121a comprising a compact beta barrel on the lower part of the outer domain in order to focus the immune response towards the b12 epitope. (Bhattacharyya et al, 2013). These were bacterially expressed, found to be partially folded, however, could bind the broadly neutralizing antibody b12 with micromolar affinity. In rabbit immunization studies sera obtained following four primes with the b122a fragment protein and two boosts with full-length gp120 showed broad neutralization of a panel of multiple viruses across different clades (Bhattacharyya et al, 2013). In the present work, These designs were further stabilised by introducing various disulphides. One of the disulphide mutants b122a1-b showed better binding to b12 compared to b122a and increased protection to protease digestion. However these are partially structured as assessed by CD. In Chapter 3 we attempted to evolve stabilized versions of b122a1-b by using a genetic selection based on antibiotic resistance described previously (Foit et al, 2009). We were successfully able to show an in-vivo stability difference between b122a and b122a1-b. From the library generated in the background of b122a1-b using random mutagenesis, a few apparently stabilized mutants were isolated. Most of these mutations were hydrophobic to polar substitutions at exposed positions while a few of the mutations were substitutions with similar side chain chemistry as in wildtype. In future studies we will measure mutant stabilities and binding affinity to b12. A set of similar fragment immunogens were also designed based on subtype C CAP210 gp120 sequences. In Chapter 4 we describe various immunization studies comprising of different sets of b12 epitope based fragment immunogens. In one study we displayed some of these immunogens on Qβ VLPs. In another study, we tested subtype C based fragment immunogens. The humoral immune response was probed in terms of generation of antibodies against the immunogens using ELISA. Neutralization activity of the sera was measured in a standard TZM-bl assay. Sera raised against these particles in rabbit immunization studies could neutralize Tier1 viruses across different subtypes. The group primed with particles displaying b122a1-b and the group primed with b122a conjugated to particle in the presence of adjuvant contained significantly higher amounts of antibodies directed towards the CD4bs than sera from the group primed with empty particles and boosted with gp120. This study demonstrates the overall utility of the particle based display approach. In immunization studies with subtype C derived fragment immunogens as primes, no significant neutralization was seen even for Tier 1 viruses. In this study, the group primed and boosted with full length gp120 performed better than other groups suggesting that antibodies elicited against regions present in these subtype C priming immunogens are non-neutralizing. One of the rational vaccine design strategies is by stabilization of native Env trimers. In previous studies, a disulfide bond was engineered between gp120 and gp41 of Env to stabilize the interactions (SOS gp140). An I559P mutation was also introduced to stabilize the native gp41 conformation in the context of disulfide engineered Env (SOSIP gp140). The purified, soluble SOSIP gp140 immunogens were trimeric and cleaved properly and are believed to be one of the closest mimics of native Env trimers. However, these immunogens have so far failed to elicit broad neutralizing responses. In Chapter 5, we use structural information derived from high resolution atomic structure of native like cleaved gp140 BG505-SOSIP, to provide an alternate strategy to form uncleaved trimeric gp140s by cyclic permutation to design molecules that mimic cleaved trimers. The structure reveals that the gp41 C-terminus is in very close proximity (~8Å) to the N-terminus of gp120 from an adjacent subunit. We have designed a cyclic permutant of gp140 from JRFL strain where the gp41 C terminus is now connected to the gp120 N-terminus with a short linker. This novel connectivity results in preservation of the native gp41 N-terminus along with a much shorter linker length than in conventional gp140. This might promote trimer folding and stabilization because of the resulting decreased magnitude of conformational entropy change during folding. The structure also reveals that the gp120 C-terminus is close to the trimer axis, and due to cyclic permutation, this becomes the new C-terminus of gp140. To further stabilize the trimeric form, we have attached a foldon trimerization domain at the C terminus. The protein has been expressed and purified from mammalian cells. The protein exists primarily as a trimer in solution as assessed by SEC-MALS. It shows better binding to broadly neutralizing antibody b12 when compared to b6, a non-neutralizing antibody. Further biophysical characterization of the protein is in progress. We have previously described design of a bacterially expressed outer domain derivative of gp120 (ODEC) that had V1/V2 and V3 loops deleted and bound CD4 (Bhattacharyya et al, 2010). To improve the initial ODEC design, three different rational design strategies were used. In the first approach, residue frequency based methods were used to design a construct named ODECConsensus. In another approach, a cyclic permutant of ODEC (CycV4OD) was designed with new N and C termini in the flexible V4 loop. In the third approach the bridging sheet (BS) region was deleted from ODEC to form ODECΔBS. In Chapter 6 we have used hydrogen deuterium exchange-mass spectrometric analysis (HDX-MS) to study conformational flexibility of these fragment immunogens. These studies revealed that all the three immunogens show reduced conformational flexibility compared to ODEC. 5-7 protons remain protected up to 2 hours whereas for ODEC, exchange completes at 20 minutes. This reduced flexibility correlates with 6-20 fold tighter VRC01 binding relative to ODEC. In rabbit immunizations, all three constructs elicit significant gp120 titers as early as week 6 in the absence of any gp120 boost whereas ODEC shows significant gp120 titers only after two gp120 boosts. Week 24 sera elicited after immunization with ODECΔBS, ODECConsensus and CycV4OD boosted with gp120 show neutralization of multiple Tier 1 viruses from subtype B and C, whereas corresponding ODEC immunized animals failed to show a neutralizing response. This study demonstrates that reduced conformational flexibility correlates with better antigenicity and an improved immunogenicity profile for these fragment immunogens. Also we have used HDX-MS studies to one of the stem based HA fragment immunogen pH1HA10-foldon described previously (Mallajosyula et al, 2014) to do peptide finger printing and find regions of protein showing increased protection to hydrogen deuterium exchange and thus derive some structural insights about this trimeric fragment immunogen. Peptide mapping experiments show that the HA stem fragment peptides are exchanging rapidly with more than 90% exchange completing by 30 s for most of the peptides. The well folded foldon trimerization domain peptide shows a very slow exchange profile. A few of the HA peptides exchange slowly with 1-2 protons exchanging after 30 s. Fast exchange seen for this fragment immunogen may be due to truncation of the stem region leading to greater solvent accessibility of the trimer interface.

No description available.

Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with partic­ular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties. The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure ade­quate vitamin deposition in the developing oocyte in the chickens. The protein is scrupu­lously conserved through evolution in terms of physico chemical and immunological char­acteristics from fish through birds to mammals, including primates. In rodents and sub­human primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Fur­ther studies with the reduced and carboxymethylated (RCM) RCP as the immunogen re­veal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines. Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic pep­tides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neu­tralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoreti­cal predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix. To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engi­neered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when ad­ministered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP. Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence. Specific antibodies were generated to this peptide as a conjugate with diphtheria tox­oid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the anti­peptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like seg­ment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.

A number of viral diseases such as Hepatitis B, small pox, measles, rubella and polio have effective vaccines to control or eradicate them. HIV-1 is a lentivirus which infects human immune cells and leads to the disease called AIDS (Acquired Immuno Deficiency Syndrome). Despite much effort since the three decades of its discovery, there is no effective vaccine against HIV-1. The envelope glycoprotein of HIV-1 is the most accessible protein on the virion surface and is essential for HIV-1 infection. Thus, this protein is the primary target for HIV-1 vaccine design. However, HIV-1 has acquired numerous immune evasive mechanisms to escape from the human immune system. Various factors such as high variability of the envelope sequence, presence of immune dominant variable loop regions, extensive glycosylation which masks conserved epitopes on the envelope, weak non-covalent interactions between gp120 and gp41 subunits of the envelope and the metastable nature of the envelope hinder the development of an effective vaccine against HIV-1. Various approaches have been carried out to design immunogens based on the envelope glycoprotein but so far none of these have succeeded in elicitation of a broad neutralizing antibody response. In chapter 1, brief descriptions of the HIV-1 epidemic, structural and genomic organization of HIV-1 along with the difficulties faced and progress in the development of an HIV-1 vaccine are described. HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers. The gp41 subunit in the native, pre-fusion trimeric Env exists in a metastable conformation and attains a stable post-fusion six helix bundle (6HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives fusion of viral and cellular membranes. The metastable nature of gp41 drives the equilibrium towards the post-fusion conformation which favours shedding of gp120 and formation of the gp41 six helix bundle remnants from the Env trimer. These dissociated products display non-neutralizing epitopes to the immune system to drive non-neutralizing antibody responses. Design and purification of Env glycoprotein in its native trimeric form is challenging due to the instability of the functional HIV-1 native Env trimer. In chapter 2, we describe our attempts to stabilize native Env trimers by incorporation of mutations at the NHR:CHR interface that disrupt the post-fusion 6HB of gp41. The mutations V570D and I573D stabilize native JRFL Env and occlude non-neutralizing epitopes to a greater extent than the previously identified I559P mutation that it is at the interface of the NHR trimers in the 6HB. The mutations prevent sCD4 induced gp120 shedding and 6HB formation. The data suggest that positions 570 and 573 are surface proximal in the native Env. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the post fusion 6HB conformation. These mutations should enhance the exposure of native Env forms to the immune system and therefore can be used to stabilize Env in a DNA vaccine format. In previous studies, a disulfide bond was engineered between gp120 and gp41 of Env to stabilize the interactions between them (SOS gp140). An I559P mutation was also introduced to stabilize the native gp41 conformation in the context of disulfide engineered Env (SOSIP gp140). The purified, soluble SOSIP gp140 immunogens were trimeric and cleaved properly. However, these immunogens failed to elicit broad neutralizing responses. The SOSIP gp140 immunogens appear to be good conformational mimics of the native trimeric Env. Thus, it is important to understand the details of the conformation and antigenic nature of SOSIP Env to further assist the design of Env immunogens in a native-like conformation. In chapter 3, we expressed JRFL-SOSIP Env on the cell surface and probed with various gp120 and gp41 specific antibodies to investigate whether this Env protein mimics the native like Env conformation. We show that introduction of a disulfide bond between gp120 and gp41 perturbs the native Env conformation, though this effect is partially alleviated by furin expression. The introduction of the V570D mutation instead of the I559P mutation partially restored the native like conformation of disulfide engineered Env. Proper cleavage of the Env to gp120 and gp41 is essential for the formation of native Env conformation. Uncleaved Env attains non-native forms and binds to non-neutralizing antibodies. To overcome inefficient cleavage problems, we co-expressed gp120 and gp41 genes on separate plasmids in mammalian cells and monitored the formation of native like Env complexes on the cell surface. We observed a fraction of native-like Env complexes on the cell surface when gp120 and gp41 with the V570D mutation are co¬expressed. We also describe the expression of Env with a self-cleavable 2A peptide between gp120 and gp41-V570D. We conclude that co-expression of gp120 and gp41 to form native like Env complexes is possible. HIV-1 Env trimeric immunogens are believed to be better immunogens than monomeric gp120. The trimeric Env immunogens designed so far, elicited marginally better neutralizing antibody response than monomeric gp120. However, these immunogens failed to elicit antibodies which could neutralize multiple primary HIV-1 isolates. Thus, it is possible that these immunogens have failed to mimic the native Env conformation. Cryo-EM and crystal structures of Env suggested that three gp120 monomers are held together at the apex of the Env trimer and the V1V2 regions of each gp120 monomer contribute to this trimeric interface. It was also shown that two broadly neutralizing antibodies (PG9 and PG16) bind to quaternary epitopes formed by V1V2 regions. Based on these observations, we hypothesized that insertion of heterologous trimerization domains into V1V2 loops might help in the formation of native like gp120 trimers. In chapter 4, two different trimerization domains (6-helix bundle and foldon trimerization domains) were inserted at the V1 loop of gp120 and C1 and C5 regions of gp120 were deleted to reduce the conformational flexibility of gp120. The resulting constructs were not trimeric and lost binding to trimer specific antibodies, PG9 and PG16. Due to their large distances between N and C-termini, these trimerization domains might have altered the local conformation of V1V2 regions and destabilized gp120 trimer formation. Interestingly, introduction of a trimerization domain (hCMP) at the C-terminus of C1 and C5 deleted gp120 (gp120-hCMP-21), led to the formation of native-like trimers which bound to both PG9 and PG16 antibodies. These results suggest that it may be difficult to trimerize gp120 by insertion of heterologous trimerization domains into the V1V2 loop and that conformational integrity of the V1V2 region is essential for the formation of trimeric gp120 interface. V1V2 regions of gp120 form quaternary epitopes on the Env trimer and are target for several broadly neutralizing antibodies. Moreover, these regions are important for the formation of the gp120 trimeric interface in the Env. In chapter 4, we show that insertion of heterologous trimerization domains at the V1 loop failed to form native like gp120 trimers. To further investigate this issue, in chapter 5, we made cyclic permutants of the gp120 molecule to create new N and C-termini at the V1 or V2 loop regions. This allowed the insertion of heterologous trimerization domains at these loop regions without affecting the folding and stability of gp120. The hCMP trimerization domain was introduced at the N-terminus of cyclically permuted gp120 (V1cyc and V2cyc). The resulting cyclic permutants were trimeric and retained binding to several broadly neutralizing antibodies. These cyclic permutants showed 10-20 fold increased binding to quaternary epitope specific neutralizing antibodies PG9 and PGT 145. CD4 binding site directed broadly neutralizing antibodies b12 and VRC01 also showed increased affinities to these cyclic permutants. Immunization of guinea pigs with cyclic permutants elicited broad neutralizing antibody response to Tier-1 and Tier-2 HIV-1 isolates with substantially higher titers than the corresponding monomeric gp120 immunogens. The data demonstrate that cyclic permutation of gp120 did not affect the structural and functional properties of gp120. It is possible to elicit broadly neutralizing sera against HIV-1 using cyclically permuted gp120 trimers in small animals. Among several proposed cryo-EM tomography structures of trimeric Env, some suggested that the V1V2 loop regions of gp120 are located close to the trimer interface while some other structures suggested that the V1V2 loop regions of gp120 are located far from the trimer axis. The present study supports Env models in which the V1V2 loops are proximal to the trimer interface. This has recently been confirmed in high resolution cryo-EM and crystal structures of HIV-1 gp140 derivatives. HIV-1 Env subunit gp120 has 50% of its molecular mass comprised of glycans which shield Env from immune recognition. Env has approximately 25 glycosylation sites of which ~4 are located in the inner domain, ~7-8 in the V1/V2 and V3 loops and the rest in the outer domain (OD). Earlier reports suggested that the glycans are indispensable for proper folding of Env and a certain level of glycan coverage is essential for maintaining infectivity of the virion. In chapter 6, we investigated the effect of removal of glycans from core gp120 on the infectivity of the HIV-1 and on the recognition of Env by various broadly neutralizing antibodies (bNAbs). We mutated the glycosylation sites in core gp120 to the second most frequent amino acids based on multiple sequence alignment. Pseudoviral infectivity assays and mammalian cell surface display experiments show that in the context of gp160, all core gp120 glycans are dispensable for viral infectivity and for recognition of bNAbs. We also show that deglycosylated molecules can serve as a starting point to re-introduce epitopes for specific glycan dependent bNAbs. Several of the constructs will also be useful for epitope mapping and Env structural characterization. Glycosylation of Env is known to inhibit binding to germline precursors of known bNAbs. In this study we show that recognition of VRC01 germline-bNAb increases substantially with the progressive loss of glycans from JRFL pseudoviruses. This work has so far resulted in the following publications (mentioned in next page).