Philip M. McKenna

Second-Generation Rabies Virus-Based Vaccine Vectors Expressing Human Immunodeficiency Virus Type 1 Gag Have Greatly Reduced Pathogenicity but Are Highly Immunogenic

ABSTRACT Rabies virus (RV) vaccine strain-based vectors show great promise as vaccines against other viral diseases such as human immunodeficiency virus type 1 (HIV-1) infection and hepatitis C, but a low residual pathogenicity remains a concern for their use. Here we describe several highly attenuated second-generation RV-based vaccine vehicles expressing HIV-1 Gag. For this approach, we modified the previously described RV vaccine vector SPBN by replacing the arginine at position 333 (R333) within the RV glycoprotein (G) with glutamic acid (E333), deleting 43 amino acids of the RV G cytoplasmic domain (CD), or combining the R333 exchange and the CD deletion. In addition, we constructed a new RV vector that expresses HIV-1 Gag from an RV transcription unit upstream of the RV phosphoprotein gene (BNSP-Gag) instead of upstream of the G gene. As expected and as demonstrated for SPBN-Gag, all vaccine vehicles were apathogenic after peripheral administration. However, the new, second-generation vaccine vectors containing modifications in the RV G were also apathogenic after intracranial infection with 105 infectious particles, and BNSP-Gag produced a 50%-reduced mortality in mice. Of note, the observed attenuation of pathogenicity did not result in either the attenuation of the humoral response against the RV G or the previously observed robust cellular response against HIV-1 Gag. These findings demonstrate that very safe and highly effective RV-based vaccines can be constructed and further emphasize their potential utility as efficacious antiviral vaccines.

Functional Human Immunodeficiency Virus Type 1 (HIV-1) Gag-Pol or HIV-1 Gag-Pol and Env Expressed from a Single Rhabdovirus-Based Vaccine Vector Genome

ABSTRACT Recombinant rabies virus (RV) vaccine strain-based vectors have been successfully developed as vaccines against other viral diseases (J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001; McGettigan et al., J. Virol. 75:8724-8732, 2001; C. A. Siler et al., Virology 292:24-34, 2002), and safety concerns have recently been addressed (McGettigan et al., J. Virol. 77:237-244, 2003). However, size limitations of the vectors may restrict their use for development of vaccine applications that require the expression of large and multiple foreign antigens. Here we describe a new RV-based vaccine vehicle expressing 4.4 kb of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol precursor Pr160. Our results indicate that Pr160 is expressed and processed, as demonstrated by immunostaining and Western blotting. Electron microscopy studies showed both immature and mature HIV-1 virus-like particles (VLPs), indicating that the expressed HIV-1 Gag Pr55 precursor was processed properly by the HIV-1 protease. A functional assay also confirmed the cleavage and functional expression of the HIV-1 reverse transcriptase (RT) from the modified RV genome. In the next step, we constructed and recovered a new RV vaccine strain-based vector expressing a chimeric HIV-189.6P RV envelope protein from an additional RV transcription unit located between the RV nucleoprotein (N) and phosphoprotein (P) in addition to HIV-1 Pr160. The 2.2-kb chimeric HIV-1/RV envelope protein is composed of the HIV-1 Env ectodomain (ED) and transmembrane domain (TD) fused to RV glycoprotein (G) cytoplasmic domain (CD), which is required for efficient incorporation of HIV-1 Env into RV particles. Of note, the expression of both HIV-1 Env and HIV-1 Pr160 resulted in an increase in the rhabdoviral genome of >55%. Both rhabdovirus-expressed HIV-1 precursor proteins were functional, as indicated by RT activity and Env-based fusion assays. These findings demonstrate that both multiple and very large foreign genes can be effectively expressed by RV-based vectors. This research opens up the possibility for the further improvement of rhabdovirus-based HIV-1 vaccines and their use to express large foreign proteins, perhaps from multiple human pathogens.

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.

Recombinant Rhabdoviruses as Potential Vaccines for HIV-1 and Other Diseases

The failure to develop vaccines to protect against important infectious diseases such as human immunodeficiency virus type I (HIV-1) or Hepatitis C virus (HCV) has increased the interest in new vaccine strategies. One of these methods is immunization with an attenuated recombinant viral vector expressing a foreign antigen, which could protect individuals from later exposure to the respective pathogen. A new method to recover a non-segmented negative-stranded RNA virus (NNSV) from cDNA was described for the first time for rabies virus (RV), a member of the rhabdovirus family. The same approach was successfully used for another rhabdovirus, vesicular stomatitis virus (VSV), and opened the possibility to use rhabdoviruses as vaccine vehicles and biomedical tools. Further research showed that the genomes of rhabdoviruses are highly flexible, easy to manipulate, and able to express large and even multiple foreign genes, and therefore are excellent vaccine candidates. In addition, it has been shown for both RV and VSV that their single surface glycoprotein G, which is responsible for attachment and fusion to the host cell, can functionally be replaced by other viral or cellular glycoproteins. This review gives an overview of the use of RV and VSV as promising new candidates in the fight against HIV-1 and other human diseases.

Distinct Mutation Pathways of Non-Subtype B HIV-1 during In Vitro Resistance Selection with Nonnucleoside Reverse Transcriptase Inhibitors

ABSTRACT Studies were conducted to investigate mutation pathways among subtypes A, B, and C of human immunodeficiency virus type 1 (HIV-1) during resistance selection with nonnucleoside reverse transcriptase inhibitors (NNRTIs) in cell culture under low-multiplicity of infection (MOI) conditions. The results showed that distinct pathways were selected by different virus subtypes under increasing selective pressure of NNRTIs. F227C and Y181C were the major mutations selected by MK-4965 in subtype A and C viruses during resistance selection. With efavirenz (EFV), F227C and V106M were the major mutations responsible for viral breakthrough in subtype A viruses, whereas a single pathway (G190A/V106M) accounted for mutation development in subtype C viruses. Y181C was the dominant mutation in the resistance selection with etravirine (ETV) in subtype A, and E138K/H221Y were the mutations detected in the breakthrough viruses from subtype C viruses with ETV. In subtype B viruses, on the other hand, known NNRTI-associated mutations (e.g., Y181C, P236L, L100I, V179D, and K103N) were selected by the NNRTIs. The susceptibility of the subtype A and B mutant viruses to NNRTIs was determined in order to gain insight into the potential mechanisms of mutation development. Collectively, these results suggest that minor differences may exist in conformation of the residues within the NNRTI binding pocket (NNRTIBP) of reverse transcriptase (RT) among the three subtypes of viruses. Thus, the interactions between NNRTIs and the residues in the NNRTIBPs of different subtypes may not be identical, leading to distinct mutation pathways during resistance selection in cell culture.

Antiviral Activity of MK-4965, a Novel Nonnucleoside Reverse Transcriptase Inhibitor

ABSTRACT Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the mainstays of therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, the effectiveness of NNRTIs can be hampered by the development of resistance mutations which confer cross-resistance to drugs in the same class. Extensive efforts have been made to identify new NNRTIs that can suppress the replication of the prevalent NNRTI-resistant viruses. MK-4965 is a novel NNRTI that possesses both diaryl ether and indazole moieties. The compound displays potency at subnanomolar concentrations against wild-type (WT), K103N, and Y181C reverse transcriptase (RT) in biochemical assays. MK-4965 is also highly potent against the WT virus and two most prevalent NNRTI-resistant viruses (viruses that harbor the K103N or the Y181C mutation), against which it had 95% effective concentrations (EC95s) of <30 nM in the presence of 10% fetal bovine serum. The antiviral EC95 of MK-4965 was reduced approximately four- to sixfold when it was tested in 50% human serum. Moreover, MK-4965 was evaluated with a panel of 15 viruses with NNRTI resistance-associated mutations and showed a superior mutant profile to that of efavirenz but not to that of etravirine. MK-4965 was similarly effective against various HIV-1 subtypes and viruses containing nucleoside reverse transcriptase inhibitor or protease inhibitor resistance-conferring mutations. A two-drug combination study showed that the antiviral activity of MK-4965 was nonantagonistic with each of the 18 FDA-licensed drugs tested vice versa in the present study. Taken together, these in vitro data show that MK-4965 possesses the desired properties for further development as a new NNRTI for the treatment of HIV-1 infection.

Highly Attenuated Rabies Virus—Based Vaccine Vectors Expressing Simian-Human Immunodeficiency Virus89.6P Env and Simian Immunodeficiency Virusmac239 Gag Are Safe in Rhesus Macaques and Protect from an AIDS-Like Disease

We analyzed the safety and immunogenicity of attenuated rabies virus vectors expressing simian-human immunodeficiency virus (SHIV)-1(89.6P) Env or simian immunodeficiency virus (SIV)(mac239) Gag in rhesus macaques. Four test macaques were immunized with both vaccine constructs, and 2 control macaques received an empty rabies vector. Seroconversion against rabies virus glycoprotein (G) and SHIV(89.6P) Env was detected after the initial immunization, but no cellular responses against SHIV antigens were observed. HIV/SIV-specific immune responses were not enhanced by boosts with the same vectors. Therefore, we constructed vectors expressing SHIV(89.6P) Env and SIV(mac239) Gag in which the rabies G was replaced with the G protein of vesicular stomatitis virus (VSV). Two years after initial immunization, a boost with the rabies-VSV G vectors resulted in SIV/HIV-specific immune responses. Upon challenge with SHIV(89.6P) test macaques controlled the infection, whereas control macaques had high levels of viremia and a profound loss of CD4(+) T cells, with 1 control macaque dying of an AIDS-like disease.

Covalently Linked Human Immunodeficiency Virus Type 1 gp120/gp41 Is Stably Anchored in Rhabdovirus Particles and Exposes Critical Neutralizing Epitopes

ABSTRACT Rabies virus (RV) vaccine strain-based vectors show significant promise as potential live-attenuated vaccines against human immunodeficiency virus type 1 (HIV-1). Here we describe a new RV construct that will also likely have applications as a live-attenuated or killed-particle immunogen. We have created a RV containing a chimeric HIV-1 Env protein, which contains introduced cysteine residues that give rise to an intermolecular disulfide bridge between gp120 and the ectodomain of gp41. This covalently linked gp140 (gp140 SOS) is fused in frame to the cytoplasmic domain of RV G glycoprotein and is efficiently incorporated into the RV virion. On the HIV-1 virion, the gp120 and gp41 moieties are noncovalently associated, which leads to extensive shedding of gp120 from virions and virus-infected cells. The ability to use HIV-1 particles as purified, inactivated immunogens has been confounded by the loss of gp120 during preparation. Additionally, monomeric gp120 and uncleaved gp160 molecules have been shown to be poor antigenic representations of virion-associated gp160. Because the gp120 and gp41 portions are covalently attached in the gp140 SOS molecule, the protein is maintained on the surface of the RV virion throughout purification. Surface immunostaining and fluorescence-activated cell sorting analysis with anti-envelope antibodies show that the gp140 SOS protein is stably expressed on the surface of infected cells and maintains CD4 binding capabilities. Furthermore, Western blot and immunoprecipitation experiments with infected-cell lysates and purified virions show that a panel of neutralizing anti-envelope antibodies efficiently recognize the gp140 SOS protein. The antigenic properties of this recombinant RV particle containing covalently attached Env, as well as the ability to present Env in a membrane-bound form, suggest that this approach could be a useful component of a HIV-1 vaccine strategy.

Immunogenicity Study of Glycoprotein-Deficient Rabies Virus Expressing Simian/Human Immunodeficiency Virus SHIV89.6P Envelope in a Rhesus Macaque

ABSTRACT Rabies virus (RV) has recently been developed as a novel vaccine candidate for human immunodeficiency virus type 1 (HIV-1). The RV glycoprotein (G) can be functionally replaced by HIV-1 envelope glycoprotein (Env) if the gp160 cytoplasmic domain (CD) of HIV-1 Env is replaced by that of RV G. Here, we describe a pilot study of the in vivo replication and immunogenicity of an RV with a deletion of G (ΔG) expressing a simian/human immunodeficiency virus SHIV89.6P Env ectodomain and transmembrane domain fused to the RV G CD (ΔG-89.6P-RVG) in a rhesus macaque. An animal vaccinated with ΔG-89.6P-RVG developed SHIV89.6P virus-neutralizing antibodies and SHIV89.6P-specific cellular immune responses after challenge with SHIV89.6P. There was no evidence of CD4+ T-cell loss, and plasma viremia was controlled to undetectable levels by 6 weeks postchallenge and has remained suppressed out to 22 weeks postchallenge.

Immunogenicity of cytopathic and noncytopathic viral vectors

ABSTRACT The impact of cytolytic versus noncytolytic viral infections on host responses is not well understood, due to limitations of the systems that have been used to address this issue. Using paired cytopathic and noncytopathic rabies viruses that differ by only two amino acids, we investigated several fundamental aspects of the immune response to these viral vectors. Greater cytopathic capacity translated into a greater degree of cross-priming to CD8+ T cells (TCD8+) and more-robust short-term humoral and cellular responses. However, long-term responses to the two viruses were similar, suggesting that direct priming drives the bulk of the TCD8+ antirabies response and that enhanced acute responses associated with greater virally mediated cellular destruction were balanced by other factors, such as prolonged antigen expression associated with noncytopathic virus. Such compensatory mechanisms may be in place to ensure comparable immunologic memories to various pathogens.