Therapeutic Advancements In Treating Zika Virus

Introduction

Zika virus (ZIKV) is a member of a family known as Flaviviridae and had been discovered upon isolation from sentinel rhesus monkeys in Africa. It has been associated with a number of outbreaks and circulating in over 25 counties around the Caribbean and Latin America. For these viruses, the intermediate hosts are monkeys, whereas the occasional hosts are humans with the transmission often being vectors from the Aedes genus. Most people infected with ZIKV are asymptomatic. An interesting group that ZIKV has been inflicting different effects on are pregnant women who have been reporting cases of congenital Zika syndrome (range of congenital disabilities) and neurological disorders such as Guillain–Barre syndrome more recently. These two factors — fetal defects and current outbreaks of the virus — have played as a catalyst for the search for effective therapeutics and vaccines to eliminate ZIKV.

Genome Organization of Zika Virus

Consisting of approximately 11,000 Kb,hthe genome of ZIKV is single-stranded positive RNA. More specifically, it consists of a single open reading frame that encodes for a polyprotein that is further cleaved into three structural proteins: capsid (C), precursor of membrane (prM), and envelope (E). E protein: plays a major role in both binding to cell surface and membrane fusion. In most vaccines, it’s the preferred antigen as it’s a primary target for neutralizing bodies. prME protein: has a role in the formation of sub-viral particles (SVPs), and it’s also used in vaccine development as they induce a neutralizing body response. C protein: has a role in interactions with lipid droplets, which is critical for membrane association and encapsidation of the viral genome. It has proven that it improves cell-mediated immune response. The open reading frame encodes for seven other non-structural proteins that are key to virus assembly, such as NS3 which cleaves both the terminal capsid and prME in conjugation with NS2B3. While the structural proteins have been prime candidates in vaccine development, some of the non-structural proteins compromise the host’s immune response as well. One example of such protein is NS4A, which inhibits the production of interferons (anti-viral molecules).

ZIKV Pathological Process

ZIKV was first identified back in 1947 and circulated within a limited parameter of land covering African and Asian countries. It remained obscure due to a limited number of reported cases until the first two outbreaks beyond Africa and Asia in Yap Island, Micronesia and French Polynesia in 2007 and 2013 respectively. Then in early 2015, Brazil witnessed a widespread epidemic of ZIKV with areas reporting seropositivity as high as 60%. For the recent cases of ZIKV infections, they are highly alerting and concerning on account of congenital disabilities (such as microcephaly) in the unborn fetus; however, the cause of the appearance of these defects remains unknown. The first link between microcephaly and ZIKV was discovered during the 2015 outbreak. The incidence of microcephaly was found to be approximately 100 folds higher than the baseline microcephaly cases in the US. In addition to that, isolation of ZIKV directly from placental tissues and infected fetuses has directly linked to the role of as a causative agent. It has not been confirmed, however, if the virus was the only causative agent. A study was done by Reynolds et al. in 2016 found that 15% of pregnant women in the US with confirmed ZIKV infection have babies with congenital disabilities associated with ZIKV.

Urgent Need for Immunization

Although ZIKV had been discovered for almost a century, there are no ZIKV-specific antivirals approved yet, and the only means of reducing the risk of transmission are by controlling mosquito vectors, making prophylactic vaccines, and preventing pregnancy in endemic areas. On account of the decline of cases reported, The World Health Organization (WHO) declared the ZIKV outbreak as no longer a Public Health Emergency of International Concern (PHEIC). Regardless, the risk of a new outbreak is not unlikely, and serious precautions should be taken, especially with the high incidence of ZIKV induced fetal disorders in pregnant women. The highest priority for women of reproductive age is the immunization of ZIKV vaccines according to the WHO Target Product Profile. Creating a vaccine safe for women of reproductive age and pregnant women to use would likely control ZIKV mediated prenatal infections and Congenital Zika Syndrome (CZS), as suggested by various models. Not only that, but a vaccine is necessary to establish a routine vaccination in areas where the vectors for this virus transmission are propagated, and this, in turn, would prevent another outbreak in the future.

Vaccine Characteristics

Any platform for a ZIKV vaccine must fulfill safety, efficacy and economical requirements to be successful. Vaccines used against other flaviviruses such as the live attenuated vaccine for Yellow Fever Virus (YFV) and the Purified Inactivated Virus (PIV) for Japanese Encephalitis Virus (JEV) are contraindicated and only given to pregnant woman if the benefits outweigh the risks. Therefore, the Zika vaccine must meet high safety standards considering the target population is women who are either pregnant or may become pregnant. The efficacy of the vaccine must also be taken into consideration as vertical transmission of ZIKV had been associated with CZS. The chance of achieving sterilizing immunity by a flavivirus vaccine is low and the titer of neutralizing antibodies that will be able to achieve this is unknown, but the desired result for the vaccine is to elicit a robust immune response that would prevent trans-placental transfer of the virus to the growing fetus.

Development of Zika Vaccine Platforms

Many vaccine candidates have been through preclinical tests, some of which made it to clinical trials between the years of 2015 and 2018. The candidates currently being under review and consideration will be reported below.

DNA Vaccines

One of the first platforms against ZIKV to be developed and most utilized the expression of prME proteins as they lead to the assembly of non-infectious sub-viral particles, which retain the structure and are antigenicity similar to native visions, in mammalian cells. Dowd et al. revealed that prME-incorporated from French Polynesian strain H/PF/2013 DNA vaccine given to mice and non-human primates provided protection against viremia. Another study done by Larocca et al. showed that mice were immunogenic against ZIKV once injected with a DNA vaccine expressing codon-optimized prME from Brazil (Strain name: BeH815744, Accession #: KU365780). Other studies in the same area done by Muthumani et al. confirm the efficacy of DNA vaccines as the experiment revealed synthetic prME constructed DNA vaccine provided protection in mice as well as non-human primates. Furthermore, IFNAR-/- mice used in the study are normally susceptible to viral infection but were protected against ZIKV after DNA vaccination. Preliminary safety shows that these vaccines are safe, and they have progressed through to clinical trials. A study done by Gaudinski et al. had tested both the efficacy and safety of VRC 5283 and VRC 5288, which are two DNA vaccines. VRC 5283 works by expressing the wild type of Zika E protein, whereas VRC 5288 expresses a chimeric E protein of Zika and Japanese encephalitis sequences. To improve particle secretion, both vaccines prM signal sequence compromised of JEV signal. By the end of phase 1, the split needle-free dose was the better candidate as the neutralizing response to VRC 5283 was superior to VRC 5288 with the presence of neutralizing bodies detected in all participants. Another study tested the efficacy of another ZIKV DNA vaccine known as GLS5700 was done by Tebas et al. . The mode of action of this vaccine is the expression of ZIKV prME regions derived from a consensus sequence, and it showed 63% development of neutralizing antibodies.

Odified mRNA Vaccines

Compared to DNA vaccines, mRNA vaccines are a safer bet as the chances of integration into the host’s genome are low. Their mode of action uses cell processes to translate the viral protein of interest. Untranslated regions at the 5’ and 3’ end confer stability in the cell as well as the replacement of uridine residues in the sequence with other modifications, and this fends off the activation of an innate immune response. Encapsulation of mRNA into lipids is also employed, for it has an advantage of aiding in intramuscular delivery, which, in turn, increases protein expression. One of the first vaccine ideas proposed after the 2015 outbreak, in fact, was the use of modified mRNA in two reports published around March 2017.

A modified mRNA vaccine was developed by Richner et al. which was encapsulated in nanoparticles made of lipids to aid in intramuscular delivery. The vaccine consisted of a signal sequence from either human IgE or JEV, prME genes of ZIKV Asian strain, and a type-1 cap. Upon the virus challenge, AG129 mice and WT C57BL/6 exhibited sterilizing immunity. Additionally, ADE role was tested and epitopes in the DII domain of the virus E protein were induced through the generation of modified mRNA vaccines. These vaccines contained mutations in the E-DII-Fusion loop region, and they have been shown to eliminate antibodies that enhanced infection of the Dengue virus. Pregnant damns were also vaccinated with the modified mRNA, and this lead to their protection against ZIKV challenge and development of congenital diseases in their offsprings. Another study was done by Similarly, Pardi et al. further confirmed the efficacy of modified mRNA vaccines encapsulated in lipid nanoparticles. The vaccine was assembled to encode for prME of ZIKV and provided immunization in mice by conferring both T and B cell response. It proved to be effective in non-human primates as it conferred strong ZIKV-specific neutralizing antibody titers after a single immunization. A recent study done in March of 2017 done by Chahal et al. discovered a nine amino acid long, highly conserved MHC-1 restricted epitope in the E protein of ZIKV capable of inducing CD8 specific T cell response in mice. The mRNA vaccine used was constructed modified from a dendrimer nanoparticle, which is different from studies discussed before, and were cloned into RNA replicon vectors that express prME protein of ZIKV.

Urified Inactivated Virus (PIV) Vaccines

DNA vaccines may provide many advantages overall such as rapid adaptation to emerging infectious agents; however, they are not always the most efficient and this was proved by a study conducted Peter et al. where PIV provided greater protection in rhesus monkeys than DNA vaccines. Furthermore, a study done by Larocca et al. in June of 2016 details the effectiveness of PIV vaccines, which was based off a Puerto Rican isolate. The vaccine was passaged through Vero cells then inactivated by formalin. Analysis of the results obtained revealed complete immunization against the virus after injection of a single dose, and even after challenging the model organism with ZIKV-BR infection. The same study entails that higher titers were achieved in intramuscular as compared to subcutaneous vaccinated mice. Later in September 2016, the effectiveness of PIV vaccines were greatly challenged. Used in two doses in conjunction with alum, rhesus monkeys vaccinated with PRVABC59 isolate were completely protected against a challenge with both the ZIKV-BR and Puerto Rican isolate. In addition to that, immunized rhesus monkeys protected mice and other monkeys from ZIKV infection by passive transfer of antibodies (dose dependent manner). A year after being given two doses of formalin, which inactivated the PRVABC59 vaccine, monkeys successfully fought off ZIKV infection. Whereas in comparison, two doses of DNA counterpart declined after a year and only provided early protection (at the peak of antibody titers).

Conclusions

The number of possible platforms that can be used for making a ZIKV vaccine, papers published, and trials conducted are far too great. The response of the research community to ZIKV has been tremendous and creates the possibility of having a viable vaccine closer than expected.