Recombinant vesicular stomatitis virus as an HIV-1 vaccine vector

Abstract

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.

Fig. 1

VSV genome organization, particle structure, and morphology. a The VSV genome encodes five major viral proteins, expressed from five discreet TUs. A single 3′ transcription promoter and genome replication promoter reside within the 50-nucleotide-long leader sequence. The antigenome replication promoter resides within the 59-nucleotide-long trailer sequence. Consensus transcription start and stop signals at the beginning and end of each TU, respectively, result in the synthesis of five VmRNAs. Genome replication occurs through a positive-sense antigenome intermediate. b The VSV particle is bullet-shaped, containing a ribonucleoprotein core surrounded by a lipid envelope that is derived from host cell plasma membrane. The N protein is closely associated with genomic RNA forming viral nucleocapsid. Viral RNA polymerase comprises a complex between the P and L proteins and is packaged inside the virus particle. G protein trimers form spike-like projections from the viral envelope that attach the virus particle to host cell receptors. The electron micrograph shows VSV particles stained with phosphotungstic acid (reprinted with permission from Rose and Whitt [87])

Fig. 2

The VSV mRNA transcription gradient. Genes proximal to the 3′ transcription promoter are transcribed more abundantly than 5′ proximal genes. The gradient of mRNA transcription arises because the RNA polymerase complex often detaches from the template at intergenic junctions and can only reinitiate transcription at the 3′ transcription promoter

Fig. 3

Rescue of rVSV from genomic cDNA. Virus rescue is initiated by cotransfecting plasmids (step 1) encoding the full-length viral genome and transacting viral polypeptides. T7 RNA polymerase, introduced into the cell by a variety of different methods, mediates RNA synthesis in the cell cytoplasm during the earliest stages of rescue, producing copies of the viral genomic RNA and transcripts encoding the transacting polypeptides (N, P, and L) needed to promote de novo assembly of a nucleocapsid (step 2). Functional nucleocapsid subsequently serves as a template for genome replication, transcription of all VmRNAs, and accumulation of viral proteins (steps 3–5) triggering ensuing events in the viral replication cycle including virus assembly (step 6) and budding (step 7)

Fig. 4

Expression of foreign antigens by prototypic rVSV vectors. Foreign antigens are expressed from a TU inserted between the virus G and L genes. The foreign gene is flanked by VSV-specific consensus transcription start and stop/polyadenylation signals

Fig. 5

Attenuation of rVSV by gene translocation. Step-wise translocation of the N gene further away from the 3′ transcription promoter leads to incremental attenuation of virus growth in vitro and in vivo. This method of attenuation relies on downregulation of N protein expression due to the 3′ to 5′ transcription gradient

Fig. 6

Attenuation of rVSV by G protein truncation. Truncation of the CT region of the rVSV G protein from 29 amino acids to either nine (CT9) or one (CT1) amino acids progressively attenuates virus growth in vitro and in vivo. It is believed that viral morphogenesis is adversely affected due to impaired interaction between shorter CTs and underlying viral core proteins

Fig. 7

Attenuation of rVSV by incorporation of M gene and ts mutations. a The VSV M gene encodes full-length M protein as well as two smaller in-frame polypeptides. Mutating the internal initiation codons from Met to Ala ablates expression of the smaller polypeptides resulting in a virus that produces minimal cytopathology during infection. These noncytopathic M gene mutations (Mncp) also attenuate virus replication. b One or more viral genes are replaced with those containing ts mutations. Viruses containing ts mutations are growth-attenuated at a defined nonpermissive temperature (typically 37–39°C) but can grow robustly at a lower, permissive temperature (32°C)

Fig. 8

Propagation defective rVSV vectors. Deletion of sequence encoding either all of the G protein (ΔG) or most of the G protein ectodomain (Gstem) results in virus that can propagate only in the presence of transcomplementing G protein. Both vectors are unable to spread beyond primary infected cells in vivo

Fig. 9

Future approaches to rVSV attenuation. Combination of attenuation strategies may further extend the range of attenuated rVSV vectors to be tested to achieve an optimum balance of safety and immunogenicity. N gene shuffles may be combined with either G protein CT truncations or Mncp mutations. Other individual gene translocations (M and G) may, on their own and in combination with other attenuation strategies, provide an optimal attenuation phenotype. Novel combinations of ts mutations may also produce rVSV vectors that can replicate well at 34–35°C, but not at core body temperature, and may be suited to intranasal vaccine delivery