HIV Env conserved element DNA vaccine alters immunodominance in macaques

ABSTRACT Sequence diversity and immunodominance are major obstacles in the design of an effective vaccine against HIV. HIV Env is a highly-glycosylated protein composed of ‘conserved’ and ‘variable’ regions. The latter contains immunodominant epitopes that are frequently targeted by the immune system resulting in the generation of immune escape variants. This work describes 12 regions in HIV Env that are highly conserved throughout the known HIV M Group sequences (Env CE), and are poorly immunogenic in macaques vaccinated with full-length Env expressing DNA vaccines. Two versions of plasmids encoding the 12 Env CE were generated, differing by 0–5 AA per CE to maximize the inclusion of commonly detected variants. In contrast to the full-length env DNA vaccine, vaccination of macaques with a combination of these 2 Env CE DNA induced robust, durable cellular immune responses with a significant fraction of CD8+ T cells with cytotoxic phenotype (Granzyme B+ and CD107a+). Although inefficient in generating primary responses to the CE, boosting of the Env CE DNA primed macaques with the intact env DNA vaccine potently augmented pre-existing immunity, increasing magnitude, breadth and cytotoxicity of the cellular responses. Fine mapping showed that 7 of the 12 CE elicited T cell responses. Env CE DNA also induced humoral responses able to recognize the full-length Env. Env CE plasmids are therefore capable of inducing durable responses to highly conserved regions of Env that are frequently absent after Env vaccination or immunologically subdominant. These modified antigens are candidates for use as prophylactic and therapeutic HIV vaccines

Identifying vulnerable sites of the HIV-1 capsid protein

Thesis (Ph.D.)--University of Washington, 2015One very challenging aspect of developing HIV vaccines and therapies is to overcome the high evolutionary rate and consequent sequence diversity of the virus. HIV is notorious for rapidly acquiring drug resistant and immune escape mutations, which allow the virus to survive and persist against antiviral drug suppression and host immune responses. I hypothesized that functionally and structurally conserved elements of the viral proteome with little-to-no tolerance to mutations would be good candidates for targets of vaccine-induced responses and antiretroviral drugs. To identify such regions in the HIV-1 capsid protein, I analyzed data from 5000 HIV capsid sequences from Genbank and the Los Alamos HIV sequence database to estimate sequence conservation and mutation frequencies. The structural location for each amino acid residue was determined based on the high-resolution X-ray crystal structure of the hexameric form of the capsid protein (the major morphological subunit of the mature HIV-1 capsid). In vitro pairwise growth competition assays were then carried out to determine the relative fitness cost of the most frequently observed mutations at capsid hexamerization interface sites and non-interface sites. Only a weak relationship between sequence conservation and the relative fitness was detected. On the other hand, the most frequently observed mutations at interface sites had larger fitness costs than the mutations at the non-interface sites, suggesting that interface sites are could be suitable targets for HIV vaccines and therapies. In addition to protein interface sites, I used homology protein modeling and two protein stability prediction methods to investigate links between changes in protein stability and the impact of mutations in the capsid protein on viral replication. I found that mutations predicted to induce large alterations in the dimerization of or the structural stability of the CA hexamer were far less likely to be found in the HIV sequence database than those not predicted to alter stability. Destabilizing mutations were also associated with deleterious phenotypes. Compared to mutation frequency, predicted protein stability was a better classifier of deleterious and non-deleterious mutations. However, a newly derived simple composite score, which takes into account both mutation frequency and the proteins stability score, performed better than both protein staibilty and mutation frequency alone. These results suggest that both sequence conservation and in silico structural stability should be used to identify potentially inactivating mutations. Utilizing optimized in vitro pairwise growth competition assays and in silico mutation and protein stability predictions, along with sequence conservation, I identified potentially important sites in the HIV-1 capsid protein that warrant further investigation as candidates for drug and CTL vaccine targets

Composite Sequence–Structure Stability Models as Screening Tools for Identifying Vulnerable Targets for HIV Drug and Vaccine Development

Rapid evolution and high sequence diversity enable Human Immunodeficiency Virus (HIV) populations to acquire mutations to escape antiretroviral drugs and host immune responses, and thus are major obstacles for the control of the pandemic. One strategy to overcome this problem is to focus drugs and vaccines on regions of the viral genome in which mutations are likely to cripple function through destabilization of viral proteins. Studies relying on sequence conservation alone have had only limited success in determining critically important regions. We tested the ability of two structure-based computational models to assign sites in the HIV-1 capsid protein (CA) that would be refractory to mutational change. The destabilizing mutations predicted by these models were rarely found in a database of 5811 HIV-1 CA coding sequences, with none being present at a frequency greater than 2%. Furthermore, 90% of variants with the low predicted stability (from a set of 184 CA variants whose replication fitness or infectivity has been studied in vitro) had aberrant capsid structures and reduced viral infectivity. Based on the predicted stability, we identified 45 CA sites prone to destabilizing mutations. More than half of these sites are targets of one or more known CA inhibitors. The CA regions enriched with these sites also overlap with peptides shown to induce cellular immune responses associated with lower viral loads in infected individuals. Lastly, a joint scoring metric that takes into account both sequence conservation and protein structure stability performed better at identifying deleterious mutations than sequence conservation or structure stability information alone. The computational sequence–structure stability approach proposed here might therefore be useful for identifying immutable sites in a protein for experimental validation as potential targets for drug and vaccine development

Relationship between sequence conservation and replication fitness.

<p>Relative fitness of all mutants evaluated as a function of database frequency of the amino acid found in the prototype COTM-CA sequence. Values shown are an average from two experiments, done in triplicate. The replication fitness of non-viable viruse is plotted as zero.</p

Viral replication fitness and growth kinetics of COTM-CA mutants in pairwise competition assays.

<p><b>A</b>) Relative fitness of the 21 viable mutants in CEMx174 cells. Values shown are an average from two experiments, with three replicates each. Error bars represent 95% confidence intervals. The dotted line represents neutral fitness. <b>B</b>) Growth kinetics of the five mutants (black lines) with substantial lower fitness compared to the COTM-CA prototype virus (gray lines). <b>C</b>) Growth kinetics of the four mutants with higher replication fitness than the prototype. Values shown are the average from one selected experiment done in triplicate. Error bar represents the standard deviation.</p

Fitness Costs of Mutations at the HIV-1 Capsid Hexamerization Interface

<div><p>The recently available x-ray crystal structure of HIV-1 capsid hexamers has provided insight into the molecular interactions crucial for the virus’s mature capsid formation. Amino acid changes at these interaction points are likely to have a strong impact on capsid functionality and, hence, viral infectivity and replication fitness. To test this hypothesis, we introduced the most frequently observed single amino acid substitution at 30 sites: 12 at the capsid hexamerization interface and 18 at non-interface sites. Mutations at the interface sites were more likely to be lethal (Fisher’s exact test p = 0.027) and had greater negative impact on viral replication fitness (Wilcoxon rank sum test p = 0.040). Among the interface mutations studied, those located in the cluster of hydrophobic contacts at NTD-NTD interface and those that disrupted NTD-CTD inter-domain helix capping hydrogen bonds were the most detrimental, indicating that these interactions are particularly important for maintaining capsid structure and/or function. These functionally constrained sites provide potential targets for novel HIV drug development and vaccine immunogen design.</p></div

Structural localization of interface mutations.

<p><b>A</b>) Two CA chains are shown as gray/black and green ribbons. The interface residues evaluated in this study are highlighted in bold and red-orange-yellow color. Other previously studied residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066065#pone.0066065-vonSchwedler1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066065#pone.0066065-Bartonova1" target="_blank">[8]</a> are highlighted in cyan-blue-purple color. The fitness impact of mutations is represented by the color shade ranging from small (yellow/cyan), moderate (orange/blue) to lethal (red/purple). <b>B</b>) Prototype residues participating in inter-domain helix capping hydrogen bonds, represented by orange lines, are shown on left. The mutations that resulted in loss of hydrogen bonds are modeled and highlighted on the right.</p

Viability and replication fitness of interface and non-interface mutants.

<p><b>A</b>) Fraction of viable and non-viable mutants in each group. <b>B</b>) Relative fitness of viable mutants at interface and non-interface sites. Values shown are an average from two experiments done in triplicate.</p

Immortalized stem cell-derived hepatocyte-like cells: An alternative model for studying dengue pathogenesis and therapy.

Suitable cell models are essential to advance our understanding of the pathogenesis of liver diseases and the development of therapeutic strategies. Primary human hepatocytes (PHHs), the most ideal hepatic model, are commercially available, but they are expensive and vary from lot-to-lot which confounds their utility. We have recently developed an immortalized hepatocyte-like cell line (imHC) from human mesenchymal stem cells, and tested it for use as a substitute model for hepatotropic infectious diseases. With a special interest in liver pathogenesis of viral infection, herein we determined the suitability of imHC as a host cell target for dengue virus (DENV) and as a model for anti-viral drug testing. We characterized the kinetics of DENV production, cellular responses to DENV infection (apoptosis, cytokine production and lipid droplet metabolism), and examined anti-viral drug effects in imHC cells with comparisons to the commonly used hepatoma cell lines (HepG2 and Huh-7) and PHHs. Our results showed that imHC cells had higher efficiencies in DENV replication and NS1 secretion as compared to HepG2 and Huh-7 cells. The kinetics of DENV infection in imHC cells showed a slower rate of apoptosis than the hepatoma cell lines and a certain similarity of cytokine profiles to PHHs. In imHC, DENV-induced alterations in levels of lipid droplets and triacylglycerols, a major component of lipid droplets, were more apparent than in hepatoma cell lines, suggesting active lipid metabolism in imHC. Significantly, responses to drugs with DENV inhibitory effects were greater in imHC cells than in HepG2 and Huh-7 cells. In conclusion, our findings suggest superior suitability of imHC as a new hepatocyte model for studying mechanisms underlying viral pathogenesis, liver diseases and drug effects