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.