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.

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).