Development of Venezuelan Equine Encephalitis Virus Vaccines

Genome and Structure

VEEV is approximately 70 nm in diameter with T=4 icosahedral symmetry. The viral RNA is encapsidated by 240 copies of the viral capsid protein bound in the N-terminus of the protein.

VEEV is a single-stranded positive-sense ribonucleic acid (RNA) virus with four nonstructural genes (nsP1, nsP2, nsP3 and nsP4) and five structural genes (capsid (C), envelope (E) 3, E2, 6k, and E1). VEEV genome has mRNA characteristics. 5' untranslated region (UTR) is capped with methyl residue present on the 7-position of capping guanosine nucleotide. A short UTR is present after the nsp4 gene sequence that has a promoter sequence for a 26S sub-genomic RNA. There are two open reading frames (ORF) in the genome. The first ORF in the 5' region translate nonstructural proteins and the second ORF in sub genomic RNA translate structural proteins. 3' end of the genome has a poly(A) tail.

Fig. 1 Organization of VEEV genome.
(Sharma A, Knollmann-Ritschel B. Viruses. 2019)

NsP1 is involved in capping the mRNA to protect the RNA from cellular nucleases. NsP2 is responsible for regulating the packaging of the viral genome into infectious virions. NsP3 interacts with host machinery to influence viral replication, while NsP4 is the RNA dependent RNA polymerase. Capsid is critical for binding to viral RNA to facilitate viral assembly. E2 is responsible for receptor binding, whereas E1 is the alphavirus fusion protein which facilitates fusion between the viral and endosomal membranes following endocytosis.

Development of Vaccine

At present, the development of VEEV vaccines has undergone different stages, such as inactivated vaccines, attenuated vaccines, and genetically engineered vaccines. However, there is currently no licensed vaccines or drugs available for human use against VEEV.

• Inactivated vaccine

Inactivated vaccines are generally safe but may induce short-lived immunity that requires frequent booster shots. Formalin inactivated TrD strain of VEEV was one of the first inactivated VEEV vaccines to be developed. However, this vaccine carries significant risks due to incomplete inactivation. In recent years, various techniques have been explored to inactivate the virus while preserving its antigenic properties. This includes the use of chemicals such as formaldehyde, binary ethylenimine (BEI), beta-propiolactone, or hydrophobic alkylating compounds like 1,5-iodonaphthylazide (INA), as well as physical methods like heat or radiation. High doses of gamma radiation can ensure a reliable, complete, and homogenous inactivation of large batches of virus particles. Therefore, this approach may fare better for FDA approval than other approaches that are currently being tested as VEEV vaccine candidates.

• Attenuated Vaccines

TC-83 is a live attenuated strain of VEEV that has been used as a vaccine in equines and humans. It was generated by passaging the virulent Trinidad Donkey (TrD) strain 83 times in guinea pig heart cells. TC-83 has limited immunogenicity, with seroconversion in only about 80% of individuals, but is fairly reactogenic (40% in vaccinated individuals). There is also concern that it may revert to the wildtype virulent form due to its attenuation relying on only two points of mutations. Other live-attenuated vaccines were engineered to improve upon the aforementioned drawbacks of TC-83. A targeted mutagenesis approach was taken to create V3526, an attenuated IAB-based VEE vaccine with a PE2 (E2 precursor protein) cleavage mutation and an E1 (Phe253Ser) suppressor substitution. V3526 showed strong immunogenicity compared with TC-83 and had the potential to be used into human. Reed et al. found that V3526 protected macaques from a potent VEEV aerosol attack, suggesting that V3526 may be a candidate vaccine strain to prevent VEEV aerosol infection.

• Subunit Vaccine

DNA constructs expressing the structural proteins of VEEV has been used as vaccine candidates. Structural genes (C-E3-E2-6K-E1) of TrD strain of VEEV cloned in mammalian expression vector pWRG7077 and administered via the epidermis using a gene-gun protected mice and macaques against an aerosol challenge with wild-type VEEV. The DNA vaccine platform expressing structural genes of VEEV is attractive from the safety standpoint as it avoids the potential of reversal of candidate vaccine platforms to the virulent type. However, low seroconversion rates, requirement of multiple doses are undesirable. Codon optimization addresses the poor immunogenicity issue, and further dose optimization for immunogenic efficacy is needed.

• Chimeric Vaccine

Chimeric vaccine aims to harness the advantageous features of multiple pathogens to enhance the immune response and provide broad protection against various diseases. To overcome the residual virulence of TC-83, structural genes were placed under the regulation of an internal ribosome entry site (IRES) of EMCV. The EMCV-IRES sequence was cloned in the subgenomic (SG) RNA, replacing the 5'UTR of the SG RNA of TC-83. The resulting TC-83 chimera (VEEV/mutSG/IRES) caused reduced mortality in neonatal mice compared to the parent TC-83 strain. Immunization with VEEV/mutSG/IRES/1 provided 80% protection against virulent VEEV subtype IC strain 3908. VEEV chimera based on a SINV backbone has also been explored as an attenuated vaccine candidate. The SINV-VEEV chimera was constructed by replacing SINV structural genes with TC-83 genes with final chimera (SIN-83) consisting of 5'UTR, nsp1-4 and 3'UTR of SINV, and structural proteins of TC-83. Stable SIN-83 chimera induced neutralizing antibody response in infant mice and protected against challenge with wild-type VEEV subtype ID and IC.

• Viral-vectored vaccines

Viral-vectored vaccines can express heterologous (different strain of the same virus) antigens and induce strong antigen-specific immune responses. Commonly used vaccine vectors include pox virus, herpes virus, adenovirus etc. The human type 5 adenovirus was used to produce E3-E2-6K of the VEEV strain TC-83 in the RAd/VEEV#3 vaccine. RAd/VEEV#3 provided almost complete protection (90%) against a homologous challenge with Trinidad donkey, but when immunized mice were challenged with heterologous strains, a variable protection (50–100%) was observed. Recently, the Eilat virus (EILV), an Alphavirus with a host range restricted to insects, was used to deliver the structural proteins C-E3-E2-6K-E1 of EEEV and VEEV. A single dose of EILV/EEEV or EILV/VEEV completely protected against homologous and heterologous challenges, respectively.