Creation of Matrix Protein Gene Variants of Two Serotypes of Vesicular Stomatitis Virus as Prime-Boost Vaccine Vectors

Abstract

To take advantage of live recombinant vesicular stomatitis viruses (rVSVs) as vaccine vectors for their high yield and for their induction of strong and long-lasting immune responses, it is necessary to make live vaccine vectors safe for use without losing their immunogenicity. We have generated safer and highly efficient recombinant VSV vaccine vectors by combining the M51R mutation in the M gene of serotype VSV-Indiana (VSV Ind ) with a temperature-sensitive mutation (tsO23) of the VSV Ind Orsay strain. In addition, we have generated two new serotype VSV-New Jersey (VSV NJ ) vaccine vectors by combining M48R and M51R mutations with G22E and L110F mutations in the M gene, rVSV NJ (G22E M48R M51R) [rVSV NJ (GMM)] and VSV NJ (G22E M48R M51R L110F) [rVSV NJ (GMML)]. The combined mutations G21E, M51R, and L111F in the M protein of VSV Ind significantly reduced the burst size of the virus by up to 10,000-fold at 37°C without affecting the level of protein expression. BHK 21 cells and SH-SY5Y human neuroblastoma cells infected with rVSV Ind (GML), rVSV NJ (GMM), and rVSV NJ (GMML) showed significantly reduced cytopathic effects in vitro at 37°C, and mice injected with 1 million infectious virus particles of these mutants into the brain showed no neurological dysfunctions or any other adverse effects. In order to increase the stability of the temperaturesensitive mutant, we have replaced the phenylalanine with alanine. This will change all three nucleotides from UUG (leucine) to GCA (alanine). The resulting L111A mutant showed the temperature-sensitive phenotype of rVSV Ind (GML) and increased stability. Twenty consecutive passages of rVSV Ind (GML) with an L111A mutation did not convert back to leucine (UUG) at position 111 in the M protein gene. V esicular stomatitis virus (VSV) is a rapidly replicating virus.Eventually humoral and cellular immune responses against VSV are elicited in the animal host, like any other viral vectors (1-3). Animals infected with VSV develop immune responses within 2 weeks and start to neutralize the VSV (1). This hinders the efficacy of boost immunizations for vaccination with the same vector. Serotype-specific antibodies against the viral surface glycoprotein G neutralize VSV. Two different serotypes of VSV, VSV-Indiana (VSV Ind ) and VSV-New Jersey (VSV NJ ), show 50% amino acid homologies in glycoprotein G (4). Antibodies raised against one serotype of VSV do not neutralize the other serotype of VSV (5). Accordingly, other investigators have used VSV Ind with its own surface glycoprotein G and VSV Ind carrying the G gene from other serotypes as a vaccine vector (6, 7).VSV causes self-limiting disease in animals such as pigs, cows, and horses, whereby vesicular lesions on the mouth, nose, teats, and hooves are cleared in a couple of weeks after the onset (8).Although it is very rare, human infections with VSV have been reported mostly in animal care workers, veterinarians, and laboratory personnel who were in close contact with the diseased animals or with the viruses (9-11)....

Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene

“…We have inserted the ZIKV PrM gene with the E gene and the E gene with msp and Gtc genes (). These rVSV Ind -GML-ZIKV E genes and rVSV NJ -GMM-ZIKV E genes have been recovered by the VSV reverse genetics system as previously described [21]. All our recombinant VSVs have been plaque-purified three consecutive times and checked the inserted foreign genes by sequencing before use for subsequent experiments.…”

“…Utilizing our rVSV platform technology [21, 22] to combine a genetically modified ZIKV envelope protein (E) gene with attenuated strains of two different serotypes of VSV, we have created our own ZIKV vaccine. We used the M gene mutant of Indiana serotype of VSV (VSV Ind -GML) and the M gene mutant New Jersey serotype (VSV NJ -GMM) to carry the genetically modified E gene of ZIKV.…”

“…There is room for an improved ZIKV vaccine to meet all the criteria for an ideal ZIKV vaccine. We have employed a genetically modified dual serotype of vesicular stomatitis virus (VSV) platform technology [21, 22] to develop a prime-boost vaccine against ZIKV. The advantages of our recombinant VSV (rVSV)-based platform technology for vaccine development are, first, a highly efficient prime-boost vaccination can be achieved with two antigenically distinct serotypes of rVSV vectors because the vector immunity against the priming Indiana serotype (VSV Ind ) will not neutralize the boosting New Jersey serotype (VSV NJ ) vector.…”

“…Thus, VSV NJ carrying the same gene of interest as rVSV Ind will provide maximum boost effects. Second, our genetically modified VSV Ind M gene mutant (rVSV Ind -GML) and genetically modified VSV NJ M gene mutant (rVSV NJ -GMM) vectors are safe [21]. Third, rVSV Ind -GML and rVSV NJ -GMM vectors carrying foreign genes replicate highly efficiently.…”

Bioreactor production of rVSV‐based vectors in Vero cell suspension cultures

“…The COVID-19 vaccine candidate rVSV Ind -msp-S F -Gtc, is a temperature-sensitive construct. It is based on a recombinant VSV Ind (GML) mutant, and shows avirulent in vivo reduced cytopathic effect in vitro at 37°C, but replicates well at 31°C (Kim et al, 2015). This attenuation was used as a strategy to further increase the safety of rVSV for its use as a human vaccine.…”