Christopher L. Parks

Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine

The acquired immunodeficiency syndrome (AIDS)-causing lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) effectively evade host immunity and, once established, infections with these viruses are only rarely controlled by immunological mechanisms. However, the initial establishment of infection in the first few days after mucosal exposure, before viral dissemination and massive replication, may be more vulnerable to immune control. Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell (TEM) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic SIVMAC239 infection early after mucosal challenge. Thirteen of twenty-four rhesus macaques receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (versus 0 of 9 DNA/Ad5-vaccinated rhesus macaques) manifested early complete control of SIV (undetectable plasma virus), and in twelve of these thirteen animals we observed long-term (≥1 year) protection. This was characterized by: occasional blips of plasma viraemia that ultimately waned; predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; no depletion of effector-site CD4+ memory T cells; no induction or boosting of SIV Env-specific antibodies; and induction and then loss of T-cell responses to an SIV protein (Vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8+ T-cell responses in the vaccine phase, and occurred without anamnestic T-cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8+ or CD4+ lymphocyte depletion and, at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations that indicate the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated TEM responses might significantly contribute to an efficacious HIV/AIDS vaccine.

Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage.

ABSTRACT Protein-encoding nucleotide sequences of the N, P, M, F, H, and L genes were determined for a low-passage isolate of the Edmonston wild-type (wt) measles virus and five Edmonston-derived vaccine virus strains, including AIK-C, Moraten, Schwarz, Rubeovax, and Zagreb. Comparative analysis demonstrated a high degree of nucleotide sequence homology; vaccine viruses differed at most by 0.3% from the Edmonston wt strain. Deduced amino acid sequences predicted substitutions in all viral polypetides. Eight amino acid coding changes were common to all vaccine viruses; an additional two were conserved in all vaccine strains except Zagreb. Comparisons made between vaccine strains indicated that commercial vaccine lots of Moraten and Schwarz had identical coding regions and were closely related to Rubeovax, while AIK-C and Zagreb diverged from the Edmonston wt along slightly different paths. These comparisons also revealed amino acid coding substitutions in Moraten and Schwarz that were absent from the closely related reactogenic Rubeovax strain. All of the vaccine viruses contained amino acid coding changes in the core components of the virus-encoded transcription and replication apparatus. This observation, combined with identification of noncoding region nucleotide changes in potential cis-acting sequences of the vaccine strains (C. L. Parks, R. A. Lerch, P. Walpita, H.-P. Wang, M. S. Sidhu, and S. A. Udem, J. Virol. 75:921–933, 2001), suggest that modulation of transcription and replication plays an important role in attenuation.

Identification and Characterization of a Regulatory Domain on the Carboxyl Terminus of the Measles Virus Nucleocapsid Protein

ABSTRACT The paramyxovirus template for transcription and genome replication consists of the RNA genome encapsidated by the nucleocapsid protein (N protein). The activity of the complex, consisting of viral polymerase plus template, can be measured with minireplicons in which the genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) antisense RNA. Using this approach, we showed that the C-terminal 24 amino acids of the measles virus N protein are dispensable for transcription and replication, based upon the truncation of N proteins used to support minireplicon reporter gene expression. Truncation at the C-terminal or penultimate amino acid 524 resulted in no change in CAT expression, whereas larger truncations spanning residues 523 to 502 were accompanied by an approximately twofold increase in basal activity. Reporter gene expression was enhanced by supplementation with the major inducible 70-kDa heat shock protein (Hsp72) for minireplicons with the N protein or the N protein truncated at position 525 or 524 but not in systems with a truncation at position 523 or 522. Naturally occurring sequence variants of the N protein with variations at positions 522 and 523 were also shown to lack Hsp72 responsiveness independent of changes in basal activity. Since these residues lie within a linear sequence predicting a direct Hsp72 interaction, N protein-Hsp72 binding reactions were analyzed by using surface plasmon resonance technology. Truncation of the C-terminal portion of the N protein by protease digestion resulted in a reduced binding affinity between Hsp72 and the N protein. Furthermore, with synthetic peptides, we established a correlation between the functional responsiveness and the binding affinity for Hsp72 of C-terminal N protein sequences. Collectively, these results show that the C-terminal 24 amino acids of the N protein represent a regulatory domain containing a functional motif that mediates a direct interaction with Hsp72.

Analysis of the Noncoding Regions of Measles Virus Strains in the Edmonston Vaccine Lineage

ABSTRACT The noncoding sequence of five Edmonston vaccine viruses (AIK-C, Moraten, Rubeovax, Schwarz, and Zagreb) and those of a low-passage Edmonston wild-type (wt) measles virus have been determined and compared. Twenty-one nucleotide positions were identified at which Edmonston wt and one or more vaccine strains differed. The location of some of these nucleotide substitutions suggests that they may influence the efficiency of mRNA synthesis, processing, and translation, as well as genome replication and encapsidation. Five nucleotide substitutions were conserved in all of the vaccine strains. Two of these were in the genomic 3′-terminal transcriptional control region and could affect RNA synthesis or encapsidation. Three were found within the 5′-untranslated region of the F mRNA, potentially altering translation control sequences. The remaining vaccine virus base changes were found in one to four vaccine strains. Their genomic localization suggests that some may modify cis-acting regulatory domains, including the Kozak consensus element of the P and M genes, the F gene-end signal, and the F mRNA 5′-untranslated sequence.

Enhanced Control of Pathogenic Simian Immunodeficiency Virus SIVmac239 Replication in Macaques Immunized with an Interleukin-12 Plasmid and a DNA Prime-Viral Vector Boost Vaccine Regimen

ABSTRACT DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent “blips” in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.

Recombinant vesicular stomatitis virus as an HIV-1 vaccine vector

Recombinant vesicular stomatitis virus (rVSV) is currently under evaluation as a human immunodeficiency virus (HIV)-1 vaccine vector. The most compelling reasons to develop rVSV as a vaccine vector include a very low seroprevalence in humans, the ability to infect and robustly express foreign antigens in a broad range of cells, and vigorous growth in continuous cell lines used for vaccine manufacture. Numerous preclinical studies with rVSV vectors expressing antigens from a variety of human pathogens have demonstrated the versatility, flexibility, and potential efficacy of the rVSV vaccine platform. When administered to nonhuman primates (NHPs), rVSV vectors expressing HIV-1 Gag and Env elicited robust HIV-1-specific cellular and humoral immune responses, and animals immunized with rVSV vectors expressing simian immunodeficiency virus (SIV) Gag and HIV Env were protected from AIDS after challenge with a pathogenic SIV/HIV recombinant. However, results from an exploratory neurovirulence study in NHPs indicated that these prototypic rVSV vectors might not be adequately attenuated for widespread use in human populations. To address this safety concern, a variety of different attenuation strategies, designed to produce a range of further attenuated rVSV vectors, are currently under investigation. Additional modifications of further attenuated rVSV vectors to upregulate expression of HIV-1 antigens and coexpress molecular adjuvants are also being developed in an effort to balance immunogenicity and attenuation.

Identification of an HIV-1 Clade A Envelope That Exhibits Broad Antigenicity and Neutralization Sensitivity and Elicits Antibodies Targeting Three Distinct Epitopes

ABSTRACT Broadly neutralizing antibodies (bNAbs) PG9 and PG16 were isolated from an International AIDS Vaccine Initiative (IAVI) Protocol G subject infected with human immunodeficiency virus type 1 (HIV-1) clade A. Both antibodies are highly potent and neutralize greater than 70% of viruses tested. We sought to begin immunogen design based on viral sequences from this patient; however, pseudoviruses prepared with 19 envelope sequences from this subject were resistant to neutralization by PG9 and PG16. Therefore, we used a bioinformatics approach to identify closely related viruses that were potentially sensitive to PG9 and PG16. A most-recent common ancestor (MRCA) sequence for the viral envelope (Env) was determined and aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database. Virus BG505.W6M.ENV.C2 (BG505) was found to have the highest degree of homology (73%) to the MRCA sequence. Pseudoviruses prepared with this Env were sensitive to neutralization with a broad panel of bNAbs, including PG9 and PG16. When expressed by 293T cells as soluble gp120, the BG505 monomer bound well to both PG9 and PG16. We further showed that a point mutation (L111A) enabled more efficient production of a stable gp120 monomer that preserves the major neutralization epitopes. Finally, we showed that an adjuvanted formulation of this gp120 protein elicited neutralizing antibodies in rabbits (following a gp120 DNA vaccine prime) and that the antisera competed with bNAbs from 3 classes of nonoverlapping epitopes. Thus, the BG505 Env protein warrants further investigation as an HIV vaccine candidate, as a stand-alone protein, or as a component of a vaccine vector.

Development of replication-competent viral vectors for HIV vaccine delivery

PURPOSE OF REVIEW To briefly describe some of the replication-competent vectors being investigated for development of candidate HIV vaccines focusing primarily on technologies that have advanced to testing in macaques or have entered clinical trials. RECENT FINDINGS Replication-competent viral vectors have advanced to the stage at which decisions can be made regarding the future development of HIV vaccines. The viruses being used as replication-competent vector platforms vary considerably, and their unique attributes make it possible to test multiple vaccine design concepts and also mimic various aspects of an HIV infection. Replication-competent viral vectors encoding simian immunodeficiency virus or HIV proteins can be used to safely immunize macaques, and in some cases, there is evidence of significant vaccine efficacy in challenge protection studies. Several live HIV vaccine vectors are in clinical trials to evaluate immunogenicity, safety, the effect of mucosal delivery, and potential effects of preexisting immunity. SUMMARY A variety of DNA and RNA viruses are being used to develop replication-competent viral vectors for HIV vaccine delivery. Multiple viral vector platforms have proven to be well tolerated and immunogenic with evidence of efficacy in macaques. Some of the more advanced HIV vaccine prototypes based on vesicular stomatitis virus, vaccinia virus, measles virus, and Sendai virus are in clinical trials.

Prevalence of specific neutralizing antibodies against Sendai virus in populations from different geographic areas: implications for AIDS vaccine development using Sendai virus vectors.

A Sendai virus (SeV) vector is being developed for delivery of an HIV immunogen. SeV is not known to cause disease in humans. Because it is genetically and antigenically related to human parainfluenza virus type 1 (hPIV-1), it is important to determine whether pre-existing hPIV-1 antibodies will affect immune responses elicited by a SeV vector-based vaccine. To quantify SeV neutralizing antibodies (NAb) in human serum, a sensitive virus neutralization assay was developed using a SeV vector encoding green fluorescent protein. Samples from 255 HIV-uninfected subjects from Africa, Europe, United States, and Japan, as well as from 12 confirmed hPIV-1-infected patients, were analyzed. SeV NAb titers did not vary significantly after serum was treated with receptor-destroying enzyme, indicating that non-specific hemagglutination inhibitors did not affect the assay sensitivity. A significant correlation was observed between hPIV-1 ELISA and SeV NAb titers. SeV NAb were detected in 92.5% subjects with a median titer of 60.6 and values ranging from 5.9- 11,324. The majority had titers

Enhanced genetic rescue of negative-strand RNA viruses: use of an MVA-T7 RNA polymerase vector and DNA replication inhibitors

A modified cDNA rescue system that improves recovery of recombinant nonsegmented, negative-strand RNA viruses from cloned DNAs is described. Rescue systems based on vaccinia virus-T7 RNA polymerase vectors have been used to derive many negative-strand viruses; however, some strains can be recalcitrant to rescue possibly because of the simultaneous replication of the vaccinia virus-T7 vector. Our goal was to engineer a system where replication of the vaccinia virus-T7 vector could be blocked, yet allow for sufficient T7 RNA polymerase expression to enable genetic rescue. To that end, a recombinant modified vaccinia virus Ankara (MVA) was engineered that contained the bacteriophage T7 gene-1 under the control of a strong early promoter that would enable T7 RNA polymerase expression in the absence of MVA DNA replication. The new T7 helper, MVAGKT7, was then utilized successfully for the genetic rescue of a measles virus minigenome and full-length cDNAs, in the presence of DNA synthesis inhibitors. In addition to blocking completely MVAGKT7 replication, AraC treatment was found to enhance minigenome-encoded gene expression and the efficiency of measles virus rescue.