Ali Azizi

Enhancing Oral Vaccine Potency by Targeting Intestinal M Cells

The immune system in the gastrointestinal tract plays a crucial role in the control of infection, as it constitutes the first line of defense against mucosal pathogens. The attractive features of oral immunization have led to the exploration of a variety of oral delivery systems. However, none of these oral delivery systems have been applied to existing commercial vaccines. To overcome this, a new generation of oral vaccine delivery systems that target antigens to gut-associated lymphoid tissue is required. One promising approach is to exploit the potential of microfold (M) cells by mimicking the entry of pathogens into these cells. Targeting specific receptors on the apical surface of M cells might enhance the entry of antigens, initiating the immune response and consequently leading to protection against mucosal pathogens. In this article, we briefly review the challenges associated with current oral vaccine delivery systems and discuss strategies that might potentially target mouse and human intestinal M cells.

Transient expression and purification of chimeric heavy chain antibodies

Monoclonal antibodies have been successfully engineered as approved therapeutics. However, their large size is considered a major factor preventing them from having a more efficient tissue penetration. As the first step to establish a possibly more efficient antibody platform, we present here transient expression, purification and characterization of six chimeric heavy chain antibodies (cHCAbs), or fusion of camelid single domain antibodies (sdAbs) to human fragment crystallizable (Fc). All six HCAbs have a MW of approximately 80 kDa, expressed well in a HEK293 expression system and have G0, G1 and G2 types of glycosylation. The transient expression also provided a very fast way to generate high milligram to low gram amount of proteins for in vitro assays and preliminary animal studies.

Innovative bioinformatic approaches for developing peptide-based vaccines against hypervariable viruses.

The application of the fields of pharmacogenomics and pharmacogenetics to vaccine design has been recently labeled ‘vaccinomics’. This newly named area of vaccine research, heavily intertwined with bioinformatics, seems to be leading the charge in developing novel vaccines for currently unmet medical needs against hypervariable viruses such as human immunodeficiency virus (HIV), hepatitis C and emerging avian and swine influenza. Some of the more recent bioinformatic approaches in the area of vaccine research include the use of epitope determination and prediction algorithms for exploring the use of peptide epitopes as vaccine immunogens. This paper briefly discusses and explores some current uses of bioinformatics in vaccine design toward the pursuit of peptide vaccines for hypervariable viruses. The various informatics and vaccine design strategies attempted by other groups toward hypervariable viruses will also be briefly examined, along with the strategy used by our group in the design and synthesis of peptide immunogens for candidate HIV and influenza vaccines.

Chemical-genetic profile analysis of five inhibitory compounds in yeast.

Background Chemical-genetic profiling of inhibitory compounds can lead to identification of their modes of action. These profiles can help elucidate the complex interactions between small bioactive compounds and the cell machinery, and explain putative gene function(s). Results Colony size reduction was used to investigate the chemical-genetic profile of cycloheximide, 3-amino-1,2,4-triazole, paromomycin, streptomycin and neomycin in the yeast Saccharomyces cerevisiae. These compounds target the process of protein biosynthesis. More than 70,000 strains were analyzed from the array of gene deletion mutant yeast strains. As expected, the overall profiles of the tested compounds were similar, with deletions for genes involved in protein biosynthesis being the major category followed by metabolism. This implies that novel genes involved in protein biosynthesis could be identified from these profiles. Further investigations were carried out to assess the activity of three profiled genes in the process of protein biosynthesis using relative fitness of double mutants and other genetic assays. Conclusion Chemical-genetic profiles provide insight into the molecular mechanism(s) of the examined compounds by elucidating their potential primary and secondary cellular target sites. Our follow-up investigations into the activity of three profiled genes in the process of protein biosynthesis provided further evidence concerning the usefulness of chemical-genetic analyses for annotating gene functions. We termed these genes TAE2, TAE3 and TAE4 for translation associated elements 2-4.

Induction of Broad Cross-Subtype-Specific HIV-1 Immune Responses by a Novel Multivalent HIV-1 Peptide Vaccine in Cynomolgus Macaques

One of the major obstacles in the design of an effective vaccine against HIV-1 is its antigenic variation, which results in viral escape from the immune system. Through a bioinformatics approach, we developed an innovative multivalent HIV-1 vaccine comprised of a pool of 176 lipidated and nonlipidated peptides representing variable regions of Env and Gag proteins. The potency and breadth of the candidate vaccine against a panel of HIV-1 subtypes was evaluated in nonhuman primate (cynomolgus macaques) and humanized mouse (HLA-A2.1) models. The results demonstrate strong immunogenicity with both breadth (humoral and cellular immunity) and depth (immune recognition of widely divergent viral sequences) against heterologous HIV-1 subtypes A–F.

Mucosal HIV vaccines: a holy grail or a dud?

The mucosal immune system appears to be a major target of the HIV infection. Therefore, a strong pre-existing anti-HIV immune response in mucosal compartments might be able to prevent HIV infection. Conflicting views regarding the mechanisms of protection at mucosal sites, inferred by the contradictory results of mucosal vaccines in human clinical trials, attests to our lack of knowledge in understanding the human mucosal immune system. In this article, we briefly review the function of innate and adaptive immune responses and discuss current strategies and potential adjuvants in designing and delivering HIV vaccines through mucosal routes.

Advances and challenges in mucosal adjuvant technology

Adjuvants play attractive roles in enhancement of immune response during vaccination; however, due to several challenges, only a limited number of adjuvants are licensed by health authorities. The lack of an effective mucosal adjuvant is even more significant as none of the licensed adjuvants revealed a strong enhancement in immune system after mucosal administration. Over the past two decades, several mucosal adjuvants have been developed to deliver antigens to the target cells in the mucosal immune system and increase specific immune responses. However, the safety and efficacy of these adjuvants for testing in human trials is still an important issue, requiring further study. In this article, we briefly review the challenges associated with most common mucosal adjuvants and discuss potential strategies for targeting the mucosal immune system.

Detection of influenza A and B neutralizing antibodies in vaccinated ferrets and macaques using specific biotin-streptavidin conjugated antibodies.

Several critical factors of an influenza microneutralization assay, utilizing a rapid biotin-streptavidin conjugated system for detecting influenza virus subtypes A and B, are addressed within this manuscript. Factors such as incubation times, amount of virus, cell seeding, sonication, and TPCK trypsin were evaluated for their ability to affect influenza virus neutralization in a microplate-based neutralization assay using Madin-Darby canine kidney (MDCK) cells. It is apparent that the amount of virus used in the assay is the most critical factor to be optimized in an influenza microneutralization assay. Results indicate that 100xTCID(50) of influenza A/Solomon Islands/03/2006 (H1N1) virus overloads the assay and results in no, to low, neutralization, in both ferret and macaque sera, respectively, whereas using 6xTCID(50) resulted in significantly improved neutralization. Conversely, strong neutralization was observed against 100xTCID(50) of B/Malaysia/2506/04 virus. In this manuscript the critical factors described above were optimized and the results indicate that the described biotin-streptavidin conjugated influenza microneutralization assay is a rapid and robust method for detecting the presence of functional, influenza virus-neutralizing antibodies.

Potency of Cell-Mediated Immune Responses to Different Combined HIV-1 Immunogens in a Humanized Murine Model

In this study, cell-mediated immune responses were evaluated in HLA-A2.1 mice that received polycistronic vector expressing HIV-1 gp120, gag and pol or single vectors expressing gp120 +gag/pol as well as recombinant structural proteins and adjuvants. Mice primed with the polycistronic DNA/CpG and boosted with the same regimen plus proteins induced a higher T-cell proliferative response to gp120. However, a very high frequency of IFN-gamma was detected in mice received the mixture of gp120 + gag/pol DNA constructs, recombinant proteins and CpG. We also measured specific CD8+T cells by intracellular cytokine staining and dimer assay in response to HIV-1 peptides specific for HLA-A2.1 in PBMCs without in vitro expansion. The group that received single gp120+gag/pol DNA constructs, recombinant proteins and CpG had a higher CD8+T cell response to the mixture of peptides in comparison with the other groups that received the polycistronic construct. The present study reveals an optimal combination of immunogens to increase immune responses against HIV-1.

A combined nucleocapsid vaccine induces vigorous SARS-CD8+ T-cell immune responses

Several studies have shown that cell-mediated immune responses play a crucial role in controlling viral replication. As such, a candidate SARS vaccine should elicit broad CD8+ T-cell immune responses. Several groups of mice were immunized alone or in combination with SARS-nucleocapsid immunogen. A high level of specific SARS-CD8+ T-cell response was demonstrated in mice that received DNA encoding the SARS-nucleocapsid, protein and XIAP as an adjuvant. We also observed that co-administration of a plasmid expressing nucleocapsid, recombinant protein and montanide/CpG induces high antibody titers in immunized mice. Moreover, this vaccine approach merits further investigation as a potential candidate vaccine against SARS.