A review on Zika vaccine development

Abstract Zika virus (ZIKV), which belongs to the Flavivirus family, is mainly transmitted via the bite of Aedes mosquitoes. In newborns, ZIKV infection can cause severe symptoms such as microcephaly, while in adults, it can lead to Guillain‒Barré syndrome (GBS). Due to the lack of specific therapeutic methods against ZIKV, the development of a safe and effective vaccine is extremely important. Several potential ZIKV vaccines, such as live attenuated, inactivated, nucleic acid, viral vector, and recombinant subunit vaccines, have demonstrated promising outcomes in clinical trials involving human participants. Therefore, in this review, the recent developmental progress, advantages and disadvantages of these five vaccine types are examined, and practical recommendations for future development are provided.


Introduction
Zika virus (ZIKV) is a mosquito-borne fla vivirus , that was isolated from a febrile rhesus monk e y in the Ugandan forest (Dick et al. 1952 , Kim andShin 2022 ).Over the last few years, there have been multiple global outbreaks of ZIKV infections, such as the one that occurred in French Polynesia between 2013 and 2014, where 28 000 individuals were infected.Approximately 440 000 to 1 300 000 people were infected with ZIKV following the outbr eak in Br azil in 2015.Fr om 2015 to January 2017, more than 800 000 confirmed and probable cases of ZIKV infection were reported in the Americas, and millions of individuals were estimated to be affected by ZIKV (Hills et al. 2017 ).Curr entl y, ZIKV infections hav e br oken out in some places from time to time, and the scope of infections has continued to expand, posing a major threat to public health (Morabito and Graham 2017 , Baker et al. 2022 ).For pregnant women, most ZIKV infections are asymptomatic; ho w e v er , mother -to-c hild tr ansmission of the virus may result in severe fetal and childhood defects such as microcephaly, lissencephaly, cortical calcifications, uveitis, and unilateral acute maculopath y.F or adults, ZIKV infection may cause Guillain-Barré syndr ome (GBS) (Oliv eir a et al. 2023, Olmo et al. 2023 ).Mor eov er, the viral RNA encodes a polypeptide precursor, and its amino terminus 1/3 produces three structural proteins, which are present in the envelope (E), premembrane/membrane (prM/M), and capsid (C) of the virion.The carboxy-terminal 2/3 of the pol ypr otein pr oduces se v en nonstructur al pr oteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (Giraldo et al. 2023 ).In viral replication and pol ypr otein pr ocessing, the NS3 pr otein is crucial and contains a helicase domain at the C-terminus and a protease domain at the N-terminus (Zhou et al. 2023 ).Furthermor e, the NS2B/NS3 pr otein plays a pr ominent r ole in vir al r eplication and maintenance of protein function (Lin et al. 2022, Chen et al. 2023 ).The E protein contains most virus virion surfaces and plays significant roles in the virus replication cycle (Chen et al. 2023, Sun et al. 2023 ).The M protein, synthesized as a prM a ppr oximatel y 164-168 aa in length, is instrumental in the assembly and folding of the E protein (Hasan et al. 2018, Sui et al. 2023 ).T hus , ZIKV candidate vaccines under development use the E and prM proteins as the primary antigens (Fialho et al. 2023 ).In this r e vie w, we summarize se v er al types of ZIKV v accines under de v elopment, including li ve-atten uated vaccines, inacti vated vaccines, n ucleic acid vaccines, vir al v ector ed v accines, and r ecombinant subunit v accines.Although these vaccines have moved from preclinical to clinical trials in various developmental stages, none have been licensed or marketed (Wang et al. 2022 ).In addition, se v er al pr actical c hallenges and difficulties in de v eloping ZIKV v accines ar e discussed.For example, the influence of pr e vious imm unity a gainst other viruses on subsequent ZIKV infection is unclear, as this immune r esponse potentiall y leads to se v er e imm une r esponses suc h as antibod y-de pendent enhancement (ADE).Furthermor e, e v aluating vaccines is challenging to evaluate vaccines when ZIKV circulation is decreasing, and vaccine efficacy cannot be estimated compr ehensiv el y because of insufficient studies on transmission.

Inactiv a ted v accines
Inactiv ated v accines ar e pr oduced by using physical heating or c hemical r ea gents to pr e v ent virus particles fr om losing their pathogenicity and retaining their antigenicity; subsequently, the remaining virus is inserted into the body of the parasitifer (Huang et al. 2023 ).Inactiv ated v accines can trigger potent antivir al r esponses against ZIKV by promoting the production of neutralizing antibodies and CD8 + and CD4 + T cells (Dutta and Langenburg 2023 ).To date, four inactivated ZIKV vaccines have been developed or completed phase I clinical trials (Table 1 ).A purified formalin-inactiv ated ZIKV v accine (ZPIV) was demonstr ated to protect BALB/c mice and nonhuman primates (NHPs) from fatal ZIKV infection.Subsequentl y, thr ough phase I, placebo-controlled, and double-blind trials on healthy adults, ZPIV was confirmed to str ongl y stim ulate the pr oduction of neutr alizing titer antibodies.Notabl y, the r eactions of participants who wer e injected with ZPIV wer e primaril y mild, causing onl y minor to moder ate r eactogenicity with only some common symptoms, such as injectionsite tenderness and headac he (Modjarr ad et al. 2018 ) In brief, inactivated vaccines have the advantage of being safe and stable during stor a ge, and they can stim ulate r obust neutralizing antibody titers in healthy individuals.Ho w e v er, due to their low imm unogenicity, inactiv ated v accines often r equir e adjuvants or multiple doses to stimulate a stronger immune response (Makhluf andShresta 2018 , Stephenson et al. 2020 ).Additionall y, v accine inactiv ation may cause epitopes to lose their effect or lead to se v er al subneutr alizing antibody r eactions (Table 2 ) (Cimica et al. 2016 ).

Liv e-atten uated vaccines
To reduce toxicity while retaining immunogenicity and stimulating v arious imm une r esponses, liv e-attenuated v accines ar e administered to combat pathogens .T here are two main approaches to de v eloping liv e-attenuated v accines: one a ppr oac h involv es the deliber ate intr oduction of specific m utations into the genome of the virus, while the other a ppr oac h entails the de v elopment of chimeric flaviviruses that express the prM/E proteins of ZIKV within the genetic fr ame w ork of y ello w fe v er virus (YFV) or Dengue virus (DENV) (Annamalai et al. 2017, Xie et al. 2018, Annamalai et al. 2019 ).The National Institute of Allergy and Infectious Diseases (NIAID) created rZIKV/D4 30-713, which is a mixture of viruses; used DENV-4 as its basis; and included the prM/E surface proteins of ZIKV as antigens .T his creation has now progressed to the first phase of clinical testing (Table 1 ) (Cimica et al. 2021 ).A vaccine containing an NS1 protein without glycosylation was tested by Richner and colleagues on mice, who found that the vaccine could effectively guard against ZIKV infectioninduced placental damage and fetal demise (Richner et al. 2017 ).In a separate study, Shan and colleagues developed a vaccine that had a deletion of 10 nucleotides within the 3'-untranslated region of the ZIKV genome; this vaccine exhibited high attenuation, imm unogenicity, and pr otection in mouse models, resulting in better T-cell responses than those elicited by the original virus Table 2. Adv anta ges and disadv anta ges of different vaccine types.

Vaccine type Ad v antages Disad v antages Principle of gener a tion
Inactiv ated v accines High safety for imm unosuppr essed people.
Better stability for stor a ge.
Low immunogenicity Need adjuvants or multiple doses to enhance immunity.
Composed of viral particles along with other pathogens that wer e cultur ed.
Li ve-atten uated vaccines Persistent immune response without adjuvants or multiple doses.
Not recommended for imm unosuppr essed people or gravidas because of potential hazards.
Reduce the virulence of a pathogen while maintaining its activity.

DNA vaccines
Better stability for stor a ge.
Better perform vaccine design by adding or deleting.
Lo w immunogenicity.Lo w ther a peutic efficacy due to the degradation of DNA.
An antigen from a pathogen is cloned and inserted into the DNA plasmid.
mRNA vaccines Provide a better safety profile because of less insertional mutations.
Low-temper atur e stor a ge owing to instability.Need to boost immunization.
Synthesized with the virtually desired sequence.
Vir al v ector ed v accines Induce stronger immune responses.
Not recommended for imm unocompr omised persons or gra vidas .
Insert genes encoding the proteins of pathogenic micr oor ganisms into the vector.(Shan et al. 2017 ).The chimeric vaccine of ZIKV prM/E and JEV SA14-14-2 de v eloped by the Beijing Institute of Microbiology and Epidemiology (BIME) protected mice and NHPs from ZIKV infection.This v accine demonstr ated good pr otection a gainst ZIKV intrauterine dissemination in mice (Li et al. 2018 ).Compared to inactiv ated v accines, liv e-attenuated v accines gener all y pr ovide fast and long-lasting immunity without requiring adjuvant or booster immunization (Pollard and Bijker 2021 ).Howe v er, for pr egnant women and imm unocompr omised individuals with underlying health conditions, the li ve-atten uated vaccine could pose a risk to their health due to its low safety and potential hazards (Table 2 ) (Yeasmin et al. 2023 ).T he volunteers r eceiv ed VRC5283 and VRC5288 and their immunogenicity and adverse reactions were assessed on the se v enth day after injection.These vaccines sho w ed good safety and tolerance in trials, with local and systemic reactogenic events that tended to be mild and moder ate, r espectiv el y.Most volunteers' local and systemic symptoms were pain and tenderness at the injection site and headache (Dowd et al. 2016, Gaudinski et al. 2018 ).Due to its favorable tolerability, VRC5283 has progressed to phase II clinical trials.Ho w e v er, additional clinical trials are still necessary to advance the development of this vaccine and assess its effectiveness in populations at risk (Lin et al. 2018 ).Another DNA candidate vaccine, GLS-5700, contained the contains plasmid pGX7201 at a 10 mg/ml concentration in sodium salt citrate buffer.In a phase I trial, Tebas et al. examined the side effects, safety, and immunogenicity of GLS-5700, and they discov er ed that most participants generated high levels of binding and neutralizing antibodies after three injections (Tebas et al. 2021 ).DNA v accines ar e stable at ambient temper atur e, r elativ el y inexpensiv e to de v elop and str aightforw ar d to manufacture, making them a cost-effective and practical option for addressing epidemics in de v eloping countries (Susc hak et al. 2017, Zou et al. 2018, Yamanaka et al. 2022 ).Ho w e v er, the major issue with DNA vaccines lies in their inadequate ability to elicit an immune response in larger animals, probably because of the inconvenience of adding the vaccine amounts used in small animal systems.Mor eov er, unform ulated and naked plasmid DNA vaccines may have low therapeutic efficacy due to the degradation of DNA (Table 2) (Hobernik and Bros 2018 ).

mRN A v accines
The oper ational mec hanism of mRNA v accines is akin to that of DNA vaccines, as they employ the host cell replication system to gener ate imm unogenic pr oteins.Moderna and its counter parts hav e de vised tw o mRN A vaccines, mRN A-1325 and mRN A-1893, whic h hav e shown encour a ging r esults in pr oviding compr ehensiv e pr otection fr om ZIKV infection in AG129 mice and rhesus monk e ys.Curr entl y, mRNA-1893 ar e in phase II trials (Table 1 ) (US Centers for Disease Control and Prevention (CDC) 2023 ).Pardi and his team formulated an mRNA vaccine with the ZIKV prM/E gene and enveloped it with lipid nanoparticles (LNPs) to create a prM/E-mRNA-LNP vaccine .T his vaccine achieved continuous immune effects after a single low-dose injection and induced a robust CD4 + response and the production of specific IgG antibodies tar geting the E pr otein in C57BL/6 and BALB/c mice .Moreo ver, the virus challenge test sho w ed no viral hematological symptoms in either model animal (Pardi et al. 2017 ).The effectiveness of humoral and cellular reactions induced b y mRN A vaccines, particularly when targeting MHC-II, may be impacted by the efficiency of nucleic acid uptake by cells.In short, while the r a pid pr oduction and flexibility of mRNA vaccines make them appealing options, importantly, they may pose potential risks due to insertional mutations, necessitating a focus on improving safety (Pardi et al. 2018, Rzymski et al. 2023, Wilder-Smith and Durbin 2023 ).Additionall y, mRNA v accines typicall y r equir e LNPs for deliv ery, m ust be stored at low temper atur es and ar e gener all y administer ed through prime-boost immunization schedules (Table 2 ) (Wollner andRichner 2021 , Yamanaka et al. 2022 ).

Viral vectored vaccines
A variety of viral vectors, including retro virus , lentivirus , and adeno-associated virus vectors, can be utilized to create vaccines that infect host cells and prompt immune responses, including humoral and cellular components such as cytotoxic T lymphocytes (CTLs) (Zhang and Zhou 2016, Mühlebach 2017, Khoshnood et al. 2022 ).These v accines ar e often used for genetic modifications and can provoke substantial innate and ada ptiv e imm une r eactions in the host or ganism.In clinical trials, adenovirus and measles v ector ed v accines hav e shown pr omise (Table 1 ) (Vemula and Mittal 2010, Capone et al. 2013, Lagunas-Rangel et al. 2017 ).For example, Ad26.ZIKV.001, a recombinant and replication-incompetent Ad26 vector encoding engineered ZIKV M and E proteins, w as sho wn to elicit potent ZIKV-specific neutr alizing r esponses with minor side effects suc h as fatigue and headac he (Salisc h et al. 2021, Shoushtari et al. 2022 ).Similarly, the prM/E proteins of ZIKV were expressed on a platform using the clinicall y a ppr ov ed r eplication-deficient c himpanzee adenovirus v ector.This v accine demonstr ated nearl y 100% pr otection a gainst ZIKV infection and facilitated the production and maintenance of antibody and T-cell responses over the long term.Additionally, the v accine pr e v ented the de v elopment of vir emia and the tr ansmission of the virus in the brain and other tissues, outperforming pr e vious v accines in this r egard (López-Camac ho et al. 2018 ).Abbink et al. administered an adenovirus vaccine to rhesus monk e ys infected with the Brazil ZIKV strain and found that neutralizing antibodies were induced in vivo, and that the virus could not be detected in the plasma (Abbink et al. 2016 ).Kim et al. further studied the effects of this vaccine on C57BL/6 mice and found that the neutralizing antibody could be detected by the plaque reduction neutralization test (PRNT) four weeks after vaccination.Furthermor e, the v accine could pr ovide passiv e pr otection a gainst ZIKV infection in juvenile pups from immunized female mice, and the degree to which IgG was transferred from the mother mice could influence the survival rate of the juvenile rat (Kim et al. 2016 ).The other two measles v ector ed v accines, MV -ZIKA and MV -ZIKA RSP, wer e demonstr ated to hav e good pr otectiv e effects on infant rat fetal models, reducing the viral load of fetuses (Nürnberger et al. 2019 ).The formulation of viral vectored vaccines may not r equir e the use of adjuvants, and in certain scenarios, booster immunization may be unnecessary.Studies show that e v en individuals or animals who hav e r eceiv ed pr e vious measles imm unization can still generate strong immune responses with measles vir al v ector v accines (Abbink et al. 2018, Cox et al. 2018, Sadoff et al. 2021 ).Ho w e v er, it is r ecommended that vir al v ector ed v accines, such as those based on measles and vaccinia viruses, be given to immunocompromised individuals or pregnant women (Ura et al. 2014, Singh et al. 2019 ).Ho w e v er, while vir al v ector ed v accines ar e efficient at stim ulating dur able cellular and humor al imm une r eactions , they ma y also trigger an anti-adeno virus imm une r esponse that can decr ease imm unogenicity in subsequent vaccinations using the same viral vector (Zhang andZhou 2016 , P er diguero et al. 2023 ).T here ma y also be challenges in using the adenovirus and vaccinia viral systems for clinical purposes due to preexisting immunity to w ar d the vectors (Table 2 ) (Cimica et al. 2021, Bifani et al. 2023 ).

Recombinant subunit vaccines
A recombinant subunit vaccine against ZIKV is created by using plasmid DNA encoding a specific gene in bacteria, yeast, or insect cells, and this a ppr oac h is considered effective and feasible for generating long-lasting protective and therapeutic immune responses (Durbin and Wilder-Smith 2017, Shukla et al. 2017, Tripathi and Shriv astav a 2018, Zhou et al. 2021, Wang et al. 2022 ).Zhang et al. created a subunit pr otein v accine that uses yeastproduced EDIII and was demonstrated to be highly efficient at producing EDIII in yeast cells while also pr e v enting the pr oduction of cr oss-r eactiv e antibodies that can enhance DENV infection (Rey et al. 2017, Zhang et al. 2019 ).Tai et al. designed a recombinant protein comprising residues 298-409 of ZIKV EDIII that can lead to the de v elopment of neutr alizing antibodies and defend a gainst ZIKV infection in pregnant mothers and fetuses (Tai et al. 2018 ).A zDIII subunit vaccine was created by Yang and colleagues using virus-like particles (VLPs) displa ying zDIII.T hese VLPs were produced using the hepatitis B cor e antigen, whic h can pr oduce humoral and cellular immunity against virus infection in mice.In comparison to DNA v accines, this pr otein-based v accine has the adv anta ge of avoiding the possibility of genome insertion or related oncogenesis .Furthermore , the safety of this protein-based v accine sur passes that of v accines based on inactiv ated viruses or viral vectors, as it eliminates the risks of incomplete inactivation and adverse reactions to the vectors in the host (Yang et al. 2017 ).After being administered a subunit vaccine candidate that contains the ZIKV E protein and two adjuvants (namely, Co-Vaccine HTTM and alum), BALB/c, Swiss Webster, and C57BL/6 mice experienced significant increases in their IgG titers as well as in the production of neutralizing antibodies to combat ZIKV infection (To et al. 2018 ).Medina et al. created two different vaccine formulations by adding the Co-Vaccine HT™ and Alhydrogel ® 85 adjuvants to the E protein through the S2 expression system in Drosophila melanogaster.Significant le v els of pr otection wer e induced by both ZIKV vaccine formulations, whereby plasma obtained from cynomolgus macaques immunized with ZIKV E effectiv el y pr otected mice a gainst vir al c hallenge and pr otected unborn c hildr en fr om the detrimental neur ovirulence of ZIKV infection (Medina et al. 2018 ).After being given a subunit vaccine composed of the first 450 aa in the N-terminal region of the E protein (E90), immunocompetent mice were exposed to ZIKV infection during fetal de v elopment and earl y infancy.The offspring of v accinated pr egnant mice wer e pr otected a gainst br ain dama ge in addition to being completel y pr otected a gainst fatal ZIKV infection in the neonatal mouse model.Mor eov er, this v accine is safe and has no potential risk of virulence or other adverse symptoms related to live vaccines (Han et  poly (I:C) as an adjuvant derived from the common sequence of the ZIKV E protein (EZIKV).The immunogenicity of this vaccine was assessed in BALB/c mice, and it was discov er ed that it trigger ed str ong specific-EZIKV imm une r esponses in terms of both humoral and cellular aspects, suggesting that it is a safe and suitable choice for preventing ZIKV infection.Compared to traditional v accines, this v accine offers striking safety and can be designed and pr oduced efficientl y with high purity.Despite being a vaccine, its low ability to trigger an immune response necessitates the use of more po w erful doses and adjuvants to achieve an effective level of protection (Table 2 ) (Amaral et al. 2020 ).To overcome this problem, coexpr essing prM/E pr oteins in insect cells or yeast cells to self-assemble into VLPs has been shown to be an effective strategy (Liu et al. 2014, Suphatrakul et al. 2015 ).VLP-based vaccines can induce the needed immune response and can be r eadil y and cost-effectiv el y manufactur ed on a large scale.Furthermor e, VLPs ar e safe because they do not include any viral genetic material and are thus noninfectious (Table 3 ) (Amaral et al. 2020 ).Garg et al. tested the immune response in BALB/c mice vaccinated with ZIKV VLPs generated using a prM-E or C-prM-E (ca psid-pr emembr ane-env elope) construct, and reported that C-prM-E VLPs could generate higher neutralizing antibody titers than prM-E VLPs, which suggested that the inclusion of C may be beneficial for ZIKV and other flaviviral VLP vaccines.In ad dition, the y also found that VLP vaccines demonstrate better effectiveness than DNA vaccines in terms of inducing a neutralizing antibody r esponse.Furthermor e, they de v eloped a m ultiv alent v accine tar geting c hikungun y a virus (CHIKV), J apanese encephalitis virus (JEV), YFV and ZIKV termed the CJaYZ vaccine based on VLPs and tested the immunogenicity of the m ultiv alent, bivalent and tetravalent vaccines in a murine model.High levels of neutralizing antibodies were detected in all groups of immunized mice.Among these, the titers of monov alent gr oups wer e the highest because they may r eceiv e the highest antigen dose.Mice that r eceiv ed biv alent and tetr av alent v accine combinations displayed a minor decrease in neutralizing antibody titers, which can be attributed to the reduction in antigen dose administered (Garg et al. 2017(Garg et al. , 2020 ) ). Boigard et al. developed ZIKV VLPs by coexpressing C-prM-E and NS2B/NS3 proteins and tested their effectiv eness a gainst ZIKV in BALB/c mice.Compared with the inactiv ated ZIKV v accine, these ZIKV VLPs could stim ulate pr omi-nentl y gr eater neutr alizing antibody titers, whic h indicated that c hemical inactiv ation ma y ha v e deleterious effects on neutr alizing epitopes within the E pr otein.Mor eov er, ZIKV VLP v accination elicited str ong neutr alizing antibody r esponses and hardl y mediated ADE in response to DENV-2, which may significantly contribute to protection against ZIKV infection.The lack of infectivity of ZIKV VLPs eliminates the need for chemical inactivation, which may influence the efficacy and safety of vaccines (Boigard et al. 2017 ).Cabr al-Mir anda et al. de v eloped a v accine candidate against ZIKV by coupling E-DIII to cucumber mosaic virus (CuMVtt) VLPs and formulating a product with a dioleoyl phosphatidylserine (DOPS) adjuvant.This vaccine could induce antibodies efficiently and neutralize the virus without predisposing the recipient to ADE with DENV infection (Cabr al-Mir anda et al. 2019 ).Curr entl y, v accines based on VLPs do not contain replicating viral genetic material and typicall y hav e excellent safety pr ofiles.Mor eov er, substantial impr ov ements in VLP production and adjuvant optimization can lead to the licensing of se v er al VLP-based vaccines to pr e v ent infectious diseases, including influenza and hepatitis A (Bovier 2008, Herzog et al. 2009, Cimica and Galarza 2017 ).

Challenges and future perspectives
Man y r esearc hers hav e attempted in r ecent years to cr eate v accines that are both effective and safe for the prevention of ZIKV infection.Various vaccine candidates have undergone preclinical testing and have shown potential for further advancement.Curr entl y, ther e ar e v arious phase I clinical trials underway for fiv e types of vaccines against ZIKV, two of which are in the phase II stage.Ho w ever, several challenges are being faced in vaccine dev elopment r egarding safety and efficacy that need to be consider ed befor e these v accines ar e licensed and used in the clinic.The first concern is the need to establish well-c har acterized pr egnancy models of ZIKV infection that are relevant to human disease and congenital Zika syndrome (CZS) prevention.Mice are often used to model human disease due to their affordability and ease of breeding and manipulation.Nevertheless, their gestation period is brief, and they possess a placental structure that is fundamentall y differ ent fr om that of NHPs and humans (Schmidt et al. 2015, Wahid et al. 2017 ).Mor eov er, the neonatal Fc receptor for IgG (FcRn) facilitates dissimilar antibody transfer across the placenta between mice and nonhuman primates(FcRn) (Roopenian and Akilesh 2007 ).NHPs are more vulnerable to ZIKV infection than model mice and exhibit placental arc hitectur es and pregnancies similar to those of humans, making them a better model.Additionall y, imm une r esponses to ZIKV infection in pr egnant baboons and rhesus macaques have been shown to be strong (Kim et al. 2019 ).Ther efor e, it is important to acknowledge the impact of existing flavivirus immunity on vaccine safety , efficacy , and immunogenicity during pregnancy.Studies have shown that the presence of neutralizing antibodies significantly contributes to pr otection a gainst ZIKV infection, but e v aluating the efficacy of vaccines for preventing CZS during pregnancy is imperative.It is crucial to note that vaccines may not be entir el y safe, nor do they impart lifelong immunity to all recipients, and challenges associated with vaccination during pregnancy, such as the immunization of vaccine platforms with new technologies, must be tested in humans (Kim et al. 2019 ).The safety and efficacy of vaccines for pregnant women and other high-risk individuals must be considered in the design process, given the clinical symptoms caused by ZIKV infection.Furthermore, as IgG elicited by vaccines and flaviviruses can be tr ansferr ed thr ough the placenta, it is crucial to consider how these antibodies will affect the health of fetuses and newborns during vaccine development.Since ZIKV and DENV shar e antigens, imm unization with a ZIKV v accine may cause cr oss-r eactiv e antibodies to worsen se v er al symptoms of DENV infection, especiall y in first-e v er v accine r ecipients (Katzelnick et al. 2020, Röbl-Mathieu et al. 2021 ).Furthermore, it is unclear how having immunity to other flaviviruses may affect a person's susceptibility to ZIKV infection, including the duration of immunity following ZIKV infection.To advance the development of vaccines for congenital and perinatal infections and safeguard ne wborns, r esearc hers m ust perform further trials in the context of mother-to-child transmission.These factors must also account for the impact of pregnancy on immunity as well as the timing of disease detection and screening (Singh et al. 2020 ).Due to the success of the coronavirus disease 2019 (COVID-19) vaccination in pregnant women, it is important to closely monitor the impact of ZIKV vaccination on pregnant women.This appr oac h entails enhancing surveillance for potential neurological and autoimmune complications and identifying an y adv erse r eactions caused by vaccination to guarantee the health of pregnant women.Mor eov er, additional time is needed to demonstrate the efficacy of vaccines in a combined population of pregnant and nonpregnant individuals (Rid and Miller 2016, Dean et al. 2019, Male 2022 ).
Vaccine r esearc h and de v elopment heavil y r el y on animal models and observational cohorts in epidemic areas, making it impossible to estimate efficacy without ongoing transmission.Although global cases of ZIKV disease have declined since 2017, in various countries and endemic r egions, tr ansmission continues to occur at low le v els, pr esenting c hallenges for v accine de v elopment and e v aluation.To addr ess this, it is necessary to de v elop contr olled human infection models for vaccine efficacy testing and to conduct further clinical experiments targeting regions severely affected worldwide (Pattnaik et al. 2020 ).Due to the significant impact of ZIKV on pregnant women, various strategies, such as enr olling onl y females who are not of c hild-bearing a ge or r equir e the use of highl y effectiv e contr aception during the study period, have been suggested to minimize the risk of infection in nonparticipants (Durbin and Whitehead 2017 ).Innov ativ e designs and ethical considerations are also vital in assessing vaccine safety and efficacy during an outbreak (Rid andMiller 2016 , Dean et al. 2019 ).

Conclusions
In gener al, fiv e types of v accines a gainst ZIKV ar e curr entl y under going clinical trials: liv e-attenuated v accines, inactiv ated v accines, nucleic acid v accines, vir al v ector ed v accines, and r ecombinant subunit v accines.Inactiv ated v accines offer conv enient pr oduction, safety, and pr eserv ation adv anta ges; ho w e v er, their imm unogenicity is gener all y lo w er than that of live attenuated vaccines, and adjuv ants ar e needed to stim ulate high-le v el imm une r esponses.DNA v accines offer stability and mass production adv anta ges, but their low immunogenicity must be addressed.To enhance their effectiv eness, v accination str ategies involving priming and boosting with different vaccine combinations ha ve pro ven successful in animal models.Compared to inactivated vaccines, vir al v ector ed v accines do not r equir e adjuv ants or booster imm unizations.Recombinant subunit vaccines stimulate long-lasting pr otectiv e and ther a peutic imm une r esponses and ar e curr entl y a practical and feasible way to develop immunity against ZIKV.
Furthermor e, de v eloping safe and effective ZIKV vaccines remains challenging for researchers and scientists.To market and pr omote awar eness, significant steps such as mass production, adjuvant selection, optimal animal model establishment, patient r ecruitment, tr ansportation methods, and imm unization sc hemes ar e necessary.Ther efor e, gov ernments should increase their efforts to assist companies in terms of policies and funds to perform further clinical trials and manufacture ZIKV vaccines for deployment.

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Table 1 .
ZIKV vaccine candidates in clinical trials.
al. 2017 " Zhu et al. 2018 ).Amaral et al. developed a recombinant protein vaccine utilizing T able 3. ZIKV -VLP-based vaccines in the developmental stage.