Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue,West Nile and Japanese encephalitis viruses

Dengue viruses (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are major global health and growing medical problems. While a live-attenuated vaccine exists since decades against the prototype flavivirus, yellow fever virus (YFV), there is an urgent need for vaccines against dengue or West Nile diseases, and for improved vaccines against Japanese encephalitis. Live-attenuated chimeric viruses were constructed by replacing the genes coding for Premembrane (prM) and Envelope (E) proteins from YFV 17D vaccine strain with those of heterologous flaviviruses (ChimeriVax™ technology). This technology has been used to produce vaccine candidates for humans, for construction of a horse vaccine for West Nile fever, and as diagnostic reagents for dengue, Japanese encephalitis, West Nile and St. Louis encephalitis infections. This review focuses on human vaccines and their characterization from the early stages of research through to clinical development. Phenotypic and genetic properties and stability were examined, preclinical evaluation through in vitro or animal models, and clinical testing were carried out. Theoretical environmental concerns linked to the live and genetically modified nature of these vaccines have been carefully addressed. Results of the extensive characterizations are in accordance with the immunogenicity and excellent safety profile of the ChimeriVax™-based vaccine candidates, and support their development towards large-scale efficacy trials and registration.     

Flavivirus vaccines

Diseases caused by flavivirus infection have been a scourge of mankind for over three centuries; with yellow fever, dengue fever and Russian spring-summer encephalitis causing epidemics resulting in thousands of fatalities. Due to the development of a safe and efficacious live-attenuated vaccine against yellow fever, this disease is no longer such a threat in countries where adequate vaccination is practised. A similarly safe and efficacious inactivated vaccine against central-European tick-borne encephalitis has also been developed and this has drastically reduced the incidence of this disease in many countries where it is endemic. In spite of these successes, the development of vaccines against other pathogenic flaviviruses, causing diseases such as dengue fever and Russian spring-summer encephalitis, have not been successful. This review attempts to summarize the development of flavivirus vaccines to date and identify areas for future improvements. Problems associated with designing flavivirus vaccines are discussed and the advantages and disadvantages of future strategies for vaccine development are considered.     

Quantitative comparison of the cross-protection induced by tick-borne encephalitis virus vaccines based on European and Far Eastern virus subtypes

Tick-borne encephalitis virus (TBEV) is a flavivirus of wide geographic distribution and the causative agent of tick-borne encephalitis (TBE), an infection of the central nervous system. TBE has the highest incidence rate in Russia, where locally produced as well as Western European vaccines for the prevention of TBE are available. The Western European vaccines are based on TBE viruses that belong to the European subtype, while the Russian vaccines are based on Far Eastern subtype viruses. The question of to which extent vaccination with a vaccine based on the European subtype is effective in protecting against the heterologous Far Eastern virus subtype - and vice versa - has not been answered conclusively. Here we immunized mice with TBE vaccines based on European and Far Eastern subtype viruses, and used an unbiased hybrid virus test system to determine cross-neutralizing antibody titers and cross-protective efficacy. All vaccines tested elicited cross-protective responses against the heterologous strains, similar to those induced against the respective homologous vaccine strains. These data, therefore, fully support the use of TBE vaccines in geographic regions where virus subtypes heterologous to the vaccine strains are prevalent.     

Recombinant chimeric Japanese encephalitis virus/tick-borne encephalitis virus is attenuated and protective in mice

Tick-borne encephalitis virus (TBEV) represents one of the most dangerous human pathogens circulating in Europe and East Asia. No effective treatment for TBEV infection currently exists, and vaccination is the primary preventive measure. Although several inactivated vaccines have been licensed, the development of novel vaccines against TBEV remains a high priority in disease-endemic countries. In the present study, a live chimeric recombinant TBEV (ChinTBEV) was created by substituting the major structural genes of TBEV for the corresponding regions of Japanese encephalitis virus (JEV) live vaccine strain SA14-14-2. The resulting chimera had a small-plaque phenotype, replicated efficiently in both mammalian and mosquito cells. The preliminary data from in vitro passaging indicated the potential for stability of ChinTBEV. ChinTBEV also exhibited significantly attenuated neuroinvasiveness in mice upon either intraperitoneal or subcutaneous inoculation in comparison with its parental TBEV. Importantly, a single immunisation with ChinTBEV elicited TBEV-specific IgG and neutralising antibody responses in a dose-dependent manner, providing significant protection against lethal TBEV challenge in mice. Taken together, the results of this proof-of-concept study indicate that ChinTBEV can be further developed as a potential vaccine candidate against TBEV infection. Moreover, the construction of this type of flavivirus chimera using a JEV vaccine strain as the genetic backbone represents a universal vaccine approach.     

Flavivirus vaccines: Virus-like particles and single-round infectious particles as promising alternatives

The genus flavivirus of the Flaviridae family includes several human pathogens, like dengue, Zika, Japanese encephalitis, and yellow fever virus. These viruses continue to be a significant threat to human health. Vaccination remains the most useful approach to reduce the impact of flavivirus fever. However, currently available vaccines can induce severe side effects or have low effectiveness. An alternative is the use of recombinant vaccines, of which virus-like particles (VLP) and single-round infectious particles (SRIP) are of especial interest. VLP consist of the virus structural proteins produced in a heterologous system that self-assemble in a structure almost identical to the native virus. They are highly immunogenic and have been effective vaccines for other viruses for over 30 years. SRIP are promising vaccine candidates, as they induce both cellular and humoral responses, as viral proteins are expressed. Here, the state of the art to produce both types of particles and their use as vaccines against flaviviruses are discussed. We summarize the different approaches used for the design and production of flavivirus VLP and SRIP, the evidence for their safety and efficacy, and the main challenges for their use as commercial vaccines.     

Flaviviruses and flavivirus vaccines

Several human-pathogenic flaviviruses (including yellow fever, dengue, Japanese encephalitis, West Nile and tick-borne encephalitis viruses) have a significant public health impact in different parts of the world and the potential of emerging in previously non-endemic regions. For some viruses, the structure of the most important immunogen, the envelope protein E, has been determined to atomic resolution by X-ray crystallography, and the architecture of virus particles has been resolved by cryo-electron microscopy. Through the combination of structural and immunological investigations, we now have a detailed understanding of the mechanisms of virus neutralization and antibody-dependent enhancement (ADE) of infectivity at a molecular level. The latter phenomenon has been proposed to play an important role in the immunopathology of severe forms of dengue virus infections (hemorrhagic dengue fever and dengue shock syndrome) and is therefore of special relevance in the context of dengue vaccines. Effective human vaccines are in use for the prophylaxis of yellow fever (live attenuated), Japanese encephalitis (live attenuated and inactivated whole virus), and tick-borne encephalitis (inactivated whole virus). Although dengue is the most important flavivirus with respect to global disease incidence, the development and use of vaccines has been hampered so far by the theoretical risk of vaccine-related adverse events such as immune enhancement of infection and the requirement to induce a long-lasting protective immune response against all four dengue serotypes simultaneously. Currently, several kinds of dengue vaccines are in development, but only one of these candidates (a chimeric dengue-yellow fever live attenuated vaccine) has reached the stage of phase 3 clinical trials.     

Engineered flavivirus vaccines control induction of crossreactive infection-enhancing and -neutralizing antibodies

Flaviviruses are important human pathogens because of their global distribution and disease severity. The high structural similarity among flaviviruses induces cross-immunity, with individual flaviviruses exhibiting crossreactive infection-enhancing and/or -neutralizing activities against other flaviviruses. Unlike neutralizing antibodies, enhancing antibodies may increase the risk of disease severity. Vaccine-induced enhancement remains a concern in the development of flavivirus vaccines. Here, we immunized mice with DNA vaccine candidates (pcJEME, pcWNME or pcZIKME) against Japanese encephalitis virus (JEV), West Nile virus (WNV) or Zika virus (ZIKV), respectively, and investigated crossreactive neutralizing and enhancing antibody activities against seven flaviviruses. pcZIKME induced higher cross-neutralization against dengue viruses than against JEV and WNV. Moreover, pcZIKME with a single amino acid substitution (D87N) showed an increase in crossreactive neutralizing activity and a decrease in enhancing activities against other flaviviruses. A similar trend was observed in pcWNME. Engineered antigen might contribute to the development of safe and effective flavivirus vaccines.     

Status of research and development of vaccines for Streptococcus pyogenes

Streptococcus pyogenes is an important global pathogen, causing considerable morbidity and mortality, especially in low and middle income countries where rheumatic heart disease and invasive infections are common. There is a number of promising vaccine candidates, most notably those based on the M protein, the key virulence factor for the bacterium. Vaccines against Streptococcus pyogenes are considered as impeded vaccines because of a number of crucial barriers to development. Considerable effort is needed by key players to bring current vaccine candidates through phase III clinical trials and there is a clear need to develop a roadmap for future development of current and new candidates.     

Immune interference in the setting of same-day administration of two similar inactivated alphavirus vaccines: Eastern equine and western equine encephalitis

We compared the effect on primary vaccination plaque-reduction neutralization 80% titers (PRNT80) responses of same-day administration (at different injection sites) of two similar investigational inactivated alphavirus vaccines, eastern equine encephalitis (EEE) vaccine (TSI-GSD 104) and western equine encephalitis (WEE) vaccine (TSI-GSD 210) to separate administration. Overall, primary response rate for EEE vaccine was 524/796 (66%) and overall primary response rate for WEE vaccine was 291/695 (42%). EEE vaccine same-day administration yielded a 59% response rate and a responder geometric mean titer (GMT) = 89 while separate administration yielded a response rate of 69% and a responder GMT = 119. WEE vaccine same-day administration yielded a 30% response rate and a responder GMT = 53 while separate administration yielded a response rate of 54% and a responder GMT = 79. EEE response rates for same-day administration (group A) vs. non-same-day administration (group B) were significantly affected by gender. A logistic regression model predicting response to EEE comparing group B to group A for females yielded an OR = 4.10 (95% CL 1.97–8.55; p = .0002) and for males yielded an OR = 1.25 (95% CL 0.76–2.07; p = .3768). WEE response rates for same-day administration vs. non-same-day administration were independent of gender. A logistic regression model predicting response to WEE comparing group B to group A yielded an OR = 2.14 (95% CL 1.22–3.73; p = .0077). We report immune interference occurring with same-day administration of two completely separate formalin inactivated viral vaccines in humans. These findings combined with the findings of others regarding immune interference would argue for a renewed emphasis on studying the immunological mechanisms of induction of inactivated viral vaccine protection.     

History of TBE vaccines

Tick borne encephalitis was a frequent viral CNS disease in Europe in the last century, with several hundred hospitalisation cases per year. The causative agent is tick-borne encephalitis virus (TBEV), a flavivirus that is transmitted by ticks. The first tissue culture derived vaccine was developed in the early 1970s and this was commercialised as a collaboration between the Institute of Virology, Vienna, Austria and Immuno AG Vienna (now Baxter Healthcare). This vaccine was highly successful during the last thirty years and its use resulted in a significant reduction in the incidence of disease in central Europe, particularly Austria. A number of modifications have been made to the manufacturing process and formulation of this vaccine over this period and a second manufacturer has also entered the market. This article describes the development and modification of these vaccines over this time frame, and illustrates the efficacy of the vaccine in preventing this severe CNS disease.     

Development and application of a flow cytometric potency assay for DNA vaccines

We have developed a rapid, reliable, and sensitive quantitative flow cytometric assay to measure the in vitro potency and stability of DNA vaccines to be delivered either by particle-mediated epidermal delivery (PMED) or by electroporation. The method involves transfecting cells with test DNA and comparing the measured antigen expression to that generated with expression from known quantities of reference material DNA. The assay was adapted for performance under Good Laboratory Practice (GLP) guidelines and was successfully utilized to perform potency testing in support of a Phase I study for two hantavirus DNA vaccines delivered by gene gun. The results from the potency assays conducted over a 24-month period using this method proved to be highly reproducible with high signal-to-noise ratios. The assay was also adapted to assess the in vitro potency and stability of a DNA vaccine for Venezuelan equine encephalitis virus that will be delivered by electroporation. Our results indicate that this assay can be readily applied to support potency and stability testing of numerous DNA vaccines delivered by various methods, including multiagent vaccines.     

Live virus vaccines based on a yellow fever vaccine backbone: Standardized template with key considerations for a risk/benefit assessment

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viruses inserted into the backbone of another virus (so-called “chimeric virus vaccines”). Many viral vector vaccines are in advanced clinical trials. The first such vaccine to be approved for marketing (to date in Australia, Thailand, Malaysia, and the Philippines) is a vaccine against the flavivirus, Japanese encephalitis (JE), which employs a licensed vaccine (yellow fever 17D) as a vector. In this vaccine, two envelope proteins (prM-E) of YF 17D virus were exchanged for the corresponding genes of JE virus, with additional attenuating mutations incorporated into the JE gene inserts. Similar vaccines have been constructed by inserting prM-E genes of dengue and West Nile into YF 17D virus and are in late stage clinical studies. The dengue vaccine is, however, more complex in that it requires a mixture of four live vectors each expressing one of the four dengue serotypes. This vaccine has been evaluated in multiple clinical trials. No significant safety concerns have been found. The Phase 3 trials met their endpoints in terms of overall reduction of confirmed dengue fever, and, most importantly a significant reduction in severe dengue and hospitalization due to dengue. However, based on results that have been published so far, efficacy in preventing serotype 2 infection is less than that for the other three serotypes. In the development of these chimeric vaccines, an important series of comparative studies of safety and efficacy were made using the parental YF 17D vaccine virus as a benchmark. In this paper, we use a standardized template describing the key characteristics of the novel flavivirus vaccine vectors, in comparison to the parental YF 17D vaccine. The template facilitates scientific discourse among key stakeholders by increasing the transparency and comparability of information. The Brighton Collaboration V3SWG template may also be useful as a guide to the evaluation of other recombinant viral vector vaccines.     

Review on flavivirus vaccine development. Proceedings of a meeting jointly organised by the World Health Organization and the Thai Ministry of Public Health, 26-27 April 2004, Bangkok, Thailand

In light of the continuous spread of human pathogenic flaviviruses, in particular the mosquito-transmitted species, vaccine development remains a high priority on the public health agenda. On 26-27 April 2004, a conference was held in Bangkok, Thailand, to review current status of flavivirus vaccine development and related issues, focussing on dengue (DEN) and Japanese encephalitis (JE). This event, co-sponsored by the World Health Organization (WHO) and the Thai Ministry of Public Health, reviewed the progress made with vaccine development, sero-epidemiological studies and other accompanying activities critical for vaccine development and vaccination. The considerable interest in and awareness of the flavivirus diseases and their prevention by public health decision makers, as well as the establishment of two dedicated programmes for dengue and Japanese encephalitis vaccine development raise hopes that new or improved vaccines will become available in the coming years.     

The suitability of yellow fever and Japanese encephalitis vaccines for immunization against West Nile virus

Seven volunteers involved in flavivirus studies have been immunized with commercial Japanese encephalitis and yellow fever vaccines JE-VAX and YF-VAX. Strong homologous and cross-reactive with West Nile virus (WNV) antibody responses with titers 1:1600 to 1:51200 were found in all donors. All donors developed high levels of yellow fever virus (YFV) and Japanese encephalitis virus (JEV) neutralizing antibodies with titers 1:50 to 1:1600 and 1:20 to 1:640, respectively, and WNV neutralizing antibodies with titers 1:10 to 1:80. In contrast, predominantly YF-specific cell-mediated immunity was detected in all immunized donors. Responses to YFV were long lasting, but the anti-JEV humoral immunity was found to decrease with time. Cross-reactive anti-WNV responses were following the same trend dropping below detectable level at 4 years post-immunization and sharply coming back after booster immunization with the JE vaccine. Thus, immunization with the commercial flavivirus JE vaccine may be beneficial for individuals at high risk of exposure to WNV, such as personnel involved in WN research.     

Novel formulations enhance the thermal stability of live-attenuated flavivirus vaccines

Thermal stability is important for the manufacture, distribution and administration of vaccines, especially in tropical developing countries, where particularly adverse field conditions exist. Current live-attenuated flavivirus vaccines exhibit relatively poor liquid stability in clinical settings, and clinicians are instructed to discard the yellow fever vaccine 1 h after reconstitution. We have identified novel combinations of excipients that greatly enhance the thermal stability of live-attenuated DEN-2 PDK-53-based flavivirus vaccine candidates. Liquid formulations comprising a sugar, albumin and a pluronic polymer minimized the loss of flavivirus infectious titer to less than 0.5 log₁₀ pfu after storage for at least 8 h at 37 °C, 7 days at room temperature or at least 11 weeks at 4 °C. Additionally, these formulations prevented reduction of viral infectivity after two freeze-thaw cycles of virus. Formulated candidate vaccines were readily lyophilized and reconstituted with minimal loss of viral titers. In mice, the formulations were safe and did not hinder the ability of the vaccine virus to generate a potent, protective immune response. These formulations provided significantly greater liquid-phase stability than has been reported previously for other flavivirus vaccine formulations. The enhanced thermal stability provided by the formulations described here will facilitate the effective distribution of flavivirus vaccines worldwide.     

Comparison of viral glycosylation using lectin blotting with Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines

We produced a Vero cell-derived inactivated Japanese encephalitis vaccine using a serum-free medium, as a substitute for the conventional mouse brain-derived Japanese encephalitis vaccine. The immunogenicity of this cell-derived vaccine was higher than that of the conventional mouse brain-derived vaccine. The results of a clinical study in humans also demonstrated higher immunogenicity of this cell-derived vaccine. No gene mutation was found in the viral structural proteins derived from Vero cells and mouse brain. So, we conducted a lectin blot analysis, assuming differing glycosylation as a cause of the higher immunogenicity in humans. The results demonstrated that vaccine reactivity varied with lectins, particularly with WGA, DBA, MAM, SSA, SBA, and GS-II. Thus, glycosylation differed with the vaccines, suggesting a possible cause of the differing immunogenicity between mice and humans.     

Immune interference after sequential alphavirus vaccine vaccinations

We compared the effect of order of administration of investigational alphavirus vaccines on neutralizing antibody response. Volunteers who received the inactivated eastern and western equine encephalitis (EEE and WEE) vaccines before live attenuated Venezuelan (VEE) vaccine had significantly lower rates of antibody response than those receiving VEE vaccine before EEE and WEE vaccines (66.7% vs. 80.6%; p = 0.026). The odds of having a VEE antibody non-response among those initially receiving EEE and WEE vaccines, adjusted for gender, were significant (odds ratio [OR] = 2.20; 95% CI = 1.2–4.1 [p = 0.0145]) as were the odds of non-response among females adjusted for group (OR = 1.81; 95% CI = 1.2–2.7 [p = 0.0037]). Antibody interference and gender effect have major implications for vaccine strategy among those receiving multiple alphavirus vaccines and those developing next generation vaccines for these threats.     

Enhancing the utility of a prM/E-expressing chimeric vaccine for Japanese encephalitis by addition of the JEV NS1 gene

Recently, we demonstrated that a single-cycle West Nile virus (WNV) named RepliVAX WN could be used to produce a chimeric Japanese encephalitis (JE) vaccine (RepliVAX JE) by replacing the WNV prM/E genes with those of JEV. Here, we tested if replacement of WNV NS1 gene in RepliVAX JE with that of JEV (producing TripliVAX JE) could produce a superior vaccine. TripliVAX JE elicited higher anti-E immunity and displayed better efficacy in mice than RepliVAX JE. Furthermore, TripliVAX JE displayed reduced immune interference caused by pre-existing anti-NS1 immunity. Thus, we propose prM/E/NS1 chimerization as a new strategy for flavivirus vaccine development.     

Tick-borne encephalitis in China: A review of epidemiology and vaccines

Tick-borne encephalitis (TBE) has been shown to be endemic in northern and western China, including the three mountain areas in Heilongjiang, Jilin, Inner Mongolia, and Xinjiang. In addition, serology evidence shows that there is human infection in south-west provinces of China, including Xizang (Tibet) and Yunnan. TBE in China is caused by the Far Eastern TBE virus subtype and there is no biphasic course for disease presentation. The majority of TBE cases in China have occurred in people who were living or working in forests. TBE vaccines became available in China soon after the virus was identified in the country and they have been used for more than 60 years to date, with different vaccine types used in different periods. Currently, an inactivated and purified whole-virus vaccine produced in a primary hamster kidney (PHK) cell line is used. Clinical trials have shown this vaccine to have higher immunogenicity and fewer adverse reactions than previous TBE vaccines. This paper provides a review on the epidemiology of TBE and the history of TBE vaccination in China.     

Anaphylaxis rates associated with COVID-19 vaccines are comparable to those of other vaccines

We retrieved data on 8940 anaphylaxis cases post-COVID-19 vaccination from the US Vaccine Adverse Event Reporting System and the European EudraVigilance from week 52/2020 through week 31/2021 and compared them with those of other vaccines. Overall, 837,830,000 COVID-19 vaccine doses were delivered in the US and Europe during the study period, for which the vaccine name was known. The mean anaphylaxis rate was estimated at 10.67 cases per 10⁶ doses of COVID-19 vaccines (range: 7.99-19.39 cases per 10⁶ doses depending on the vaccine). COVID-19 vaccines ranked fifth in reported anaphylaxis rates, behind rabies, tick-borne encephalitis, measles-mumps-rubella-varicella, and human papillomavirus vaccines (70.77, 20, 19.8, and 13.65 cases per 10⁶ vaccine doses, respectively). COVID-19 vaccines are within the range of anaphylaxis rates reported across several common vaccines in these two passive reporting systems. These data should be communicated to reassure the general population about the safety profile of COVID-19 vaccines.