No description available.

The human papilloma virus (HPY) infection has a causal association with cervical intraepithelial neoplasia (CIN) and cervical cancer. However, pre-malignant or malignant transformation is not always observed with HPY infection. lILA molecules are important in the regulation of the immune response to foreign antigens. The role of genetic variation at the HLA class II loci (DR and DQ) in CIN was investigated in 176 British Caucasian patients and 420 controls (normal cervical cytology and negative for HPY 16, 18, 31 and 33). HLA DQB 1 *03 typing was performed by a novel polymerase chain reactionrestriction fragment length polymorphism method (A-RFLP). The technique uses PCR to mutate the first base of codon 40 (DQ alleles) from T to G to create an artificial restriction site for an enzyme, MluI, which distinguishes DQB 1 *03 from other alleles and is confirmed by digestion of amplified DNA with Mlul. Further HLA DR-DQ typing was performed by PCR DNA amplification and oligonucleotide probe typing. HPY types (16, 18, 31 & 33) were detected by using type-specific oligonucleotide primers and PCR. The alleles of the DQB 1 *03, DRB 1 *04 and DRB 1 * 11 groups were strongly associated with susceptibility to CIN. Specifically the haplotypes DRB 1 *040 I-DQB 1 *0301 and DRBl*1101-DQB1*0301 were significant and indicated susceptibility. The DQBl*03 locus was more contributory to this association than the DRB 1 loci. A weak protective effect was shown for the haplotype DRB 1 *0 10 I-DQB 1 *0501. Positive correlation was also observed for HPY-positive CIN, suggesting that specific HLA alleles may be important in determining the immune response to HPY antigens and the risk for CIN after HPY infection. Immunoaffinity purification of the susceptibility and protective HLA ~ molecules was performed and the naturally processed peptides were eluted and sequenced by Edman degradation. The data obtained was used for motif prediction of HPY 16 E6, E7, Ll and L2 sequences that may be capable of binding to these HLA molecules. Motif prediction as well as the binding affinity of predicted peptide motifs for HLA D RB 1 *0401 and DRB 1 *0 10 1 was accomplished using the published data' on the naturally bound peptide sequences bound to these HLA molecules. The results revealed significant differences in both the number and binding affinity of the HPV 16 derived peptides to the protective and susceptibility HLA molecules. These results should help in the rational design of vaccines against HPV.

<p>Large-scale, in vitro vaccine screening is an expensive and slow process, while rational vaccine design is faster and cheaper. As opposed to the emperical ways to design vaccines in biology laboratories, rational vaccine design models the structure of vaccines with computational approaches. Building an effective predictive computer model requires extensive knowledge of the process or phenomenon being modelled. Given current knowledge about the steps involved in immune system responses, computer models are currently focused on one or two of the most important and best known steps; for example: presentation of antigens by major histo-compatibility complex (MHC) molecules. In this step, the MHC molecule selectively binds to some peptides derived from antigens and then presents them to the T-cell. One current focus in rational vaccine design is prediction of peptides that can be bound by MHC.<p>Theoretically, predicting which peptides bind to a particular MHC molecule involves discovering patterns in known MHC-binding peptides and then searching for peptides which conform to these patterns in some new antigenic protein sequences. According to some previous work, Hidden Markov models (HMMs), a machine learning technique, is one of the most effective approaches for this task. Unfortunately, for computer models like HMMs, the number of the parameters to be determined is larger than the number which can be estimated from available training data.<p>Thus, heuristic approaches have to be developed to determine the parameters. In this research, two heuristic approaches are proposed. The rst initializes the HMM transition and emission probability matrices by assigning biological meanings to the states. The second approach tailors the structure of a fully connected HMM (fcHMM) to increase specicity. The effectiveness of these two approaches is tested on two human leukocyte antigens(HLA) alleles, HLA-A*0201 and HLAB* 3501. The results indicate that these approaches can improve predictive accuracy. Further, the HMM implementation incorporating the above heuristics can outperform a popular prole HMM (pHMM) program, HMMER, in terms of predictive accuracy.

<p>Large-scale, in vitro vaccine screening is an expensive and slow process, while rational vaccine design is faster and cheaper. As opposed to the emperical ways to design vaccines in biology laboratories, rational vaccine design models the structure of vaccines with computational approaches. Building an effective predictive computer model requires extensive knowledge of the process or phenomenon being modelled. Given current knowledge about the steps involved in immune system responses, computer models are currently focused on one or two of the most important and best known steps; for example: presentation of antigens by major histo-compatibility complex (MHC) molecules. In this step, the MHC molecule selectively binds to some peptides derived from antigens and then presents them to the T-cell. One current focus in rational vaccine design is prediction of peptides that can be bound by MHC.<p>Theoretically, predicting which peptides bind to a particular MHC molecule involves discovering patterns in known MHC-binding peptides and then searching for peptides which conform to these patterns in some new antigenic protein sequences. According to some previous work, Hidden Markov models (HMMs), a machine learning technique, is one of the most effective approaches for this task. Unfortunately, for computer models like HMMs, the number of the parameters to be determined is larger than the number which can be estimated from available training data.<p>Thus, heuristic approaches have to be developed to determine the parameters. In this research, two heuristic approaches are proposed. The rst initializes the HMM transition and emission probability matrices by assigning biological meanings to the states. The second approach tailors the structure of a fully connected HMM (fcHMM) to increase specicity. The effectiveness of these two approaches is tested on two human leukocyte antigens(HLA) alleles, HLA-A*0201 and HLAB* 3501. The results indicate that these approaches can improve predictive accuracy. Further, the HMM implementation incorporating the above heuristics can outperform a popular prole HMM (pHMM) program, HMMER, in terms of predictive accuracy.

The HIV-1 envelope glycoprotein trimer (Env) is covered by an extensive array of glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints that limit the actions of glycan processing enzymes, such that multiple under-processed structures remain on specific locations. These oligomannose-type glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, perhaps paradoxically, target it. In multiple studies, bNAbs have been shown to be capable of providing passive protection from viral challenge, making Env a focus of antibody-mediated vaccine design. Here, the development of a workflow for the semi-quantitative, site-specific N-glycosylation analysis of a soluble recombinant, native-like trimer mimic (BG505 SOSIP.664) is reported. The resulting data reveal a mosaic of dense clusters on the outer domain of Env and allow mapping the extremes of simplicity and diversity of glycan processing. Although individual sites usually minimally affect the global integrity of the glycan shield, examples are identified of how deleting certain glycans can subtly influence neutralization by bNAbs that bind at distant sites. Env is a trimer of heterodimers of gp120 and gp41, which is generated by cleavage of an endogenous protease. In this thesis, the detailed effect of protease cleavage on glycan processing is examined by comparing the site-specific N-glycosylation profiles of the native-like trimer mimic to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. Trimer-associated glycan remodeling forms a localized subdomain of the native mannose patch. Furthermore, the glycosylation analysis of further Env immunogens – a glycan-depleted trimer and a flexibly-linked, uncleaved trimer (both based on BG505 SOSIP.664) – provides important insights into the robustness of the HIV-1 glycan shield and the Env maturation pathway. Overall, this thesis reveals how structural constraints shape Env glycosylation and the network of bNAb-targeted glycans that should be preserved on recombinant vaccine candidates.

The multifunctional signaling hub p62 is well recognized as a ubiquitin sensor and a selective autophagy receptor. As a ubiquitin sensor, p62 promotes NFκB activation by facilitating TRAF6 ubiquitination and aggregation. As a selective autophagy receptor, p62 sorts ubiquitinated substrates including p62 itself for lysosome-mediated degradation. p62 plays crucial roles in myriad cellular processes including DNA damage response, aging/senescence, infection and immunity, chronic inflammation, and cancerogenesis, dependent on or independent of autophagy. Targeting p62-mediated autophagy may represent a promising strategy for clinical interventions of different cancers. In this review, we summarize the transcriptional and post-translational regulation of p62, and its mechanistic roles in cancers, with the emphasis on its roles in regulation of DNA damage response and its connection to the cGAS-STING-mediated antitumor immune response, which is promising for cancer vaccine design.

Hepatitis C virus (HCV) is a major global pathogen that infects an estimated 170 million people worldwide, and for which currently no vaccine is available. HCV is a highly diverse viral pathogen and exists as 6 major genotypes sharing only 75% sequence homology; developing a vaccine that is cross-reactive between genotypes is a major challenge. Defining immune responses that target different HCV genotypes will facilitate pan-genotypic T cell vaccine development. HCV genotype 3 (gt3) is now the most common infecting genotype in the United Kingdom and large parts of Asia; however, data regarding the T cell antigenic targets of this genotype is very limited. In this thesis, HCV gt3 specific T cell targets were defined in acute, chronic and spontaneously resolved infection: in chronic gt3 infection, T cell responses were low in magnitude and narrowly focused in specificity, similar to those previously reported for gt1; in contrast, resolved infection was associated with a higher magnitude and broader specificity of CD4+ and CD8+ T cell responses across the genome. Overall, T cell specificity in gt3 infection was markedly different to that previously described for gt1, confirming that sequence differences between genotypes result in distinct immunological profiles. Previous work from our laboratory demonstrated that, though T cell responses induced by a potent T cell vaccine containing HCV gt1b non-structural regions do target epitopes dominant in natural infection, induced T cells show limited cross-reactivity against other genotypes. In this thesis, it was assessed whether T cells primed in natural gt3 infection are able to recognize viral sequence variants at dominant epitopes, which would make these potential targets in cross-reactive vaccine design. For seven gt3-specific T cell epitopes identified here as dominant, major sequence variability was observed within and between genotypes, and limited T cell cross-reactivity observed against identified viral variants. This suggests that regions frequently targeted in natural infection may not serve as attractive targets for cross-reactive vaccine design. These results informed the subsequent design of a cross-reactive vaccine based on fragments of HCV that are conserved between genotypes. A generic algorithm was developed to define viral regions conserved between major HCV genotypes (for 1a/1b, 1/3a, 1-6), and these were joined to form immunogens between 819 and 1543 AA long. Possible artificial, non-HCV epitopes formed by junctions were identified using online epitope prediction servers, and abrogated through the insertion of 2-6 amino acid linkers. To address the concern that conserved regions may not be immunogenic, epitopes described in natural HCV infection were mapped on HCV sequences, showing that conserved segments are well populated with epitopes; additionally, strong binding peptides were predicted for conserved segments using online epitope prediction programs, suggesting potential in vivo immunogenicity. In conclusion, HCV T cell specificity is distinct between genotypes, with limited T cell cross-reactivity between viral variants. Leading from this result, vaccine immunogens were designed entirely based on conserved viral regions. This work paves the way for future studies of novel HCV immunogens based on conserved viral segments between genotypes.

<p>One promising HIV-1 vaccine target is the membrane-proximal external region (MPER) of viral gp41. MPER is poorly immunogenic, however, the two rare neutralizing antibodies (NAbs), 2F5 and 4E10, bind to MPER with great neutralizing ability. Although their neutralizing mechanism represents a promising framework for the design of new HIV-1 liposomal vaccine candidates, this mechanism remains poorly understood. It is known that 2F5 and 4E10 are required to first associate with HIV-1 lipids before binding to the target MPER antigen, however, little is known about how lipid membranes contribute to NAb-antigen binding. To this end we have developed model membrane systems to study NAb and antigen lipid interactions. </p><p>We first created a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol monolayers, which mimic the surface chemical properties of lipid membranes. Next, we focused on mimicking the lipid phase organization (i.e., domain formation) of native membranes by using supported lipid bilayers (SLBs). We used simple SLB compositions to model the liquid-disordered (Ld) and gel phases. To model the HIV-1 envelope, we used a complex SLB composition that contains an Ld and liquid-ordered (Lo) phase. To reliably create model HIV-1 SLBs, we developed an SLB formation technique that uses amphipathic, &#945;-helical peptides as a catalyst to generate complex SLBs that have a high cholesterol content and contain multiple lipid types. For all SLB surfaces we used atomic force microscopy (AFM) to visualize membrane domains, antigen presentation, and antibody-membrane interactions.</p><p>Results from experiments using thiol surfaces showed that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies. This supports the hypothesis that these NAbs embed into the hydrophobic membrane core. Our results demonstrate that 2F5/4E10 do not interact with the highly ordered gel and Lo domains in the SLB but exclusively bind to the Ld phase. This suggests that 2F5/4E10 require low membrane order and weak lateral lipid-lipid interactions to insert into the hydrophobic membrane interior. Thus, vaccine liposomes that primarily contain an Ld phase are more likely to elicit the production of lipid reactive, 2F5- and 4E10-like antibodies, compared to liposomes that contain an Lo or gel phase. In the context of liposomal antigen presentation, our results show that the presence of the MPER656 antigen can severely limit the Ld area available for antibody interactions. Subsequently, this reduces the amount of MPER656 that is accessible for 2F5/4E10 binding, since MPER656 preferentially localizes to the Ld area. If Ld forming lipid components are used in vaccine liposomes, it is important to ensure that the presence of antigen does not inhibit large-scale Ld formation.</p> / Dissertation

No description available.

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.