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HIV-1 is the causative agent for AIDS and has been a major focus of research for the past two decades. Though there is a combination therapy in place known as the “Highly Active Anti-Retroviral Therapy” (HAART), its usefulness is confounded by the generation of escape mutants, a host of side effects, and its prohibitive cost. The most useful alternative would be the prevention of infection by vaccination. Vaccine research has been focused on the use of recombinant protein sub-units of the virus or combinations thereof to elicit a neutralizing response against the virus. These approaches have mostly resulted in a failure to generate broadly cross reactive neutralizing response against primary strains of the virus. The work reported herein is aimed at designing a rigidified version of gp120/gp120 derivatives and understanding the scope of the various antigenic regions in gp120 in generating a neutralization response. Chapter one discusses some general features of the virus and the immune system. The general nature of AIDS, its spread and its immunological characteristics are also described in this chapter. Chapter two discusses the design and NMR structural analysis of gp120 bridging sheet peptide mimics in methanol and water. The structure of gp120 can be loosely divided into two domains (the outer domain and the inner domain) that are linked together by a discontinuous four stranded antiparallel beta sheet known as the bridging sheet. The bridging sheet is known to overlap with the coreceptor binding site of gp120 and hence is a suitable target for designing virus-entry inhibitors. 17b, a neutralizing antibody isolated from an infected individual, is known to bind to this region of gp120. Our aim in this part of the work was to design a four stranded antiparallel beta sheet, based on the sequence of the bridging sheet, that would contain most of the residues involved in 17b binding. NMR and CD studies confirmed that the peptide was well structured in methanol but the structure was largely lost on addition of aqueous solvent. A small population of the peptide was found to be well-folded in aqueous solution. Chapter three discusses the design and characterization of a gp120-CD4D12 single chain. It is well known that the conformation of gp120 changes upon binding CD4 to expose cryptic epitopes, known as CD4i epitopes. In this work we report the generation of a single chain gp120-CD4 construct that has the cryptic epitopes exposed. The construct bound to 17b, a conformation specific antibody against the bridging sheet of gp120, a cryptic epitope, as well as a non-covalent complex of gp120:CD4D12. There was also very insignificant secondary structural change in gp120 upon complex formation with CD4D12 as observed by CD spectroscopy. Immunological studies with DNA and protein vaccination in guinea-pigs indicated that though 17b like antibodies are generated after immunization, they did not contribute towards the neutralization of primary isolates of the virus. It was also observed that it was the anti-CD4D12 antibodies that were responsible for the neutralization by the sera. These studies indicated towards the inability of the bridging sheet to generate effective neutralization response in case of vaccination with gp120/CD4 complexes. Chapter four discusses the design of a mimic of the gp120/CD4 complex. Since it was seen from our previous work that gp120/CD4 complexes generate a large fraction of antiCD4 antibodies and hence are unsuitable for vaccination purposes, we generated a construct with the minimal binding region of CD4. The small fragment of CD4 spanning from 21st residue to 64th residue was inserted in the V1/V2 loop of gp120. The insertion site was designed based on the region of gp120 closest to this fragment and capable of tolerating insertions. This protein did not bind to 17b as well as gp120/CD4 complex but showed a higher binding compared to full length gp120. Further immunological characterization with this protein revealed that it was not capable of generating neutralizing antibodies against the virus. Chapter five discusses the design and execution of a SPR based solution phase competition experiment to find the solution phase binding constant of CD4 and CD4 analogs to gp120. A major problem during the analysis of binding data obtained by SPR is the accurate determination of Rmax, a parameter needed to obtain an accurate equilibrium dissociation constant. In this chapter we have developed a binary as well as a ternary solution phase SPR based assay to accurately determine a solution phase equilibrium binding constant. The binding constants were determined for gp120 binding to CD4D12 and other CD4 analogs. To confirm the validity of the assay, a control antigen:antibody interaction whose equilibrium dissociation constant has been determined by other methods has been used as a test case. Chapter six discusses the design and characterization of V3 peptides inserted in the loop regions of E. coli Thioredoxin (Trx). Trx has earlier been used to display random peptide libraries between the 33rd and the 34th residue. We have constructed three constructs where the peptide has been inserted between the 33rd and 34th residue, between the 74th and 75th residue and between the 84th and 85th residue. The insertion between 74th and 75th position (74V3Trx) was found to be superior to the other two and would be a suitable alternative for display of a random peptide library. The binding of these constructs to 447-52D, a V3 peptide specific antibody was characterized. These were also characterized immunologically, and 74V3Trx was found to generate weakly neutralizing activity against the MN strain of HIV-1. Competition experiments with 447-52D with these sera indicated that there were antibodies generated that could compete out 447-52D binding to gp120 but not in sufficient concentration to provide broad neutralization. Appendix 1 discusses the rational design of disulfides to stabilize proteins based on the analysis of naturally occurring disulfides. In our attempts to design a rigidified version of gp120 we had designed disulfides in gp120 based on its crystal structure. Many of these were disulfides that would span antiparallel adjacent strands. In order to improve the design principles, we analyzed naturally occurring disulfides that span antiparallel adjacent strands and characterized them in terms of their positional preference in a beta sheet. It was found that these disulfides mostly occur on edge strands and are found exclusively between non-hydrogen bonded registered pairs of adjacent antiparallel strands. Mutagenesis on Thioredoxin was performed to verify our results. It was found that disulfides designed between the non-hydrogen bonded pairs of antiparallel strands could significantly stabilize the protein whereas the ones between hydrogen bonded pairs destabilized the protein.

Since, its discovery over three decades ago, HIV has wrecked havoc worldwide. According to the UNAIDS report 2011, at present 34 million people is living with HIV and AIDS vaccine with broadly neutralizing activity still remains elusive. The envelope glycoproteins on the virion surface, is the most accessible component to the host immune system and therefore is targeted for vaccine design. However, the virus has employed various strategies to avoid the host immune response. The extremely high rate of mutations, extensive glycosylation of the envelope glycoprotein, conformational flexibility of the envelope, has made all the efforts aimed to design a broadly neutralizing immunogen futile. In Chapter1, we briefly discuss about the structural and genomic organization of the HIV-1 along with various strategies the virus has employed to evade the immune system. We also present the progress and failures encountered in the past three decades, on the way to design protective HIV vaccine and inhibitors. On the host cell surface, HIV-1 glycoprotein gp120 binds to the cell surface receptor CD4 and leads to the fusion of viral and host cellular membranes. CD4 is present on the surface of T-lymphocytes. It consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1−D4. sCD4 has been used as an entry inhibitor against HIV-1. However, this molecule could not neutralize primary isolates of the virus. Previously, from our lab, we had reported the design and characterization of a construct consisting of the first two domains of CD4 (CD4D12), that binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192−16202]. In Chapter 2, we describe the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. All the mutations in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC50s as 4-domain CD4. These stabilized derivatives of human CD4 are useful starting points for the design of other more complex viral entry inhibitors. 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. One group of cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface and the other group claimed that the V1V2 loop region is far from the apex of the trimer. To further investigate the position of the V1V2 region, in the native envelope trimer, in Chapter 3, we describe the design and characterization of cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, 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 than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with ~15-fold higher affinity than 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. HIV-1 envelope (env) protein gp120 has approximately 25 glycosylation sites of which ~4 are located in the inner domain, ~7-8 in the V1/V2 and V3 variable loops and the rest in the outer domain (OD) of gp120. These glycans shield env from recognition by the host immune system and are believed to be indispensable for proper folding of gp120 and viral infectivity. However, there is no detailed study that describes whether a particular potential n-linked glycan is indispensable for folding of gp120.Therefore, in Chapter 4, using rationally designed mutations and yeast surface display (YSD), we show that glycosylation is not essential for the correct in vivo folding of OD alone or OD in the context of core gp120. Following randomization of the remaining four glycosylation sites, we isolated a core gp120 mutant, which contained a single inner domain glycan and retained yeast surface expression and broadly neutralizing antibody (bNAb) binding. Thus demonstrates that most gp120 glycans are dispensable for folding in the absence of gp41. However in the context of gp160, we show that all core gp120 glycans are dispensable for folding, recognition of bNAbs and for viral infectivity. We also show that deglycosylated molecules can serve as a starting point to re-introduce epitopes for specific glycan dependent bNAbs. Several of these 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. Hence the present results inform immunogen design, clarify the role of glycosylation in gp120 folding and illustrate general methodology for design of glycan free, folded protein derivatives. On the virion surface env glycoproteins gp120 and gp41 interact via non-covalent interactions and form trimers of heterodimers. Upon binding cell surface receptor CD4 and co-receptor CCR5/CXCR4, gp120 and gp41 undergo a lot of conformational changes, which ultimately lead to the fusion of viral and cellular membranes by formation of six-helix bundle in gp41. High resolution structural information is available for core gp120 and post-fusion six-helix bundle conformation of gp41. However, the structural information about the native gp120:gp41 interface in the native trimer is lacking. In Chapter 5, we describe the design and characterization of various single chain derivatives of gp120 inner doamin and gp41. Among the designed constructs, gp41-id2b is folded but is a mixture of dimer and monomer under native conditions. To facilitate, trimer formation, two trimerization domains (h-CMP and Foldon) were individually fused to the N-terminus of gp41-id2b to generate h-CMP-gp41-id2b and Foldon-gp41-id2b. Although, these molecules were proteolytically more stable than gp41-id2b, they did not form trimer under native conditions. All the single chain derivatives were designed based on the crystal structure of gp120, which was devoid of C1 and C5 domains (PDBID 1G9M). A new set of constructs to mimic the native gp120:gp41 interface will be designed and characterized based on the recently solved crystal structure of gp120 with the C1 and C5 domains (PDBID 3JWD and 3JWO). Helix-helix interactions are fundamental to many biological signals and systems, found in homo- or hetero-multimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six helix bundle (6HB) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Yeast surface two-hybrid (YS2H) system is a platform, which is designed to detect protein-protein interactions occurring through a secretory pathway. In Chapter 6, we describe the use of aYS2H system, to reconstitute 6HB complex on the yeast surface and delineate the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for facile characterization of hetero-oligomeric interactions and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils. However, using this YS2H platform, the native hetero-oligomeric complex of gp120 and gp41 could not be captured. In Appendix 1, we report cloning, expression and purification of PΔGgp120 and ΔGgp120 from methylotrophic yeast Pichia pastoris. PΔGgp120 was purified as a secreted protein. However, in electrophoretic analyses the molecule ran as a heterogeneous smear. Further optimization of the purification protocol and biophysical characterizations of this molecule will be performed in future. In Appendix 2, gp41 variants were expressed on the yeast cell surface as a C-terminally fused protein and its interaction with externally added gp120 was monitored by FACS. The surface expression of the gp41 constructs was poor and they did not show any interaction with gp120.