Biodefense Vaccines, Vaccines for Emerging Infectious Diseases, and Coalition for Epidemic Preparedness Innovations (CEPI)

Venezuelan Equine Encephalitis Virus

The two VEE vaccines used under INDs—included the live-attenuated product, TC-83; and a formalin-inactivated product, C-84;—both derive from the same lineage. The live-attenuated TC-83 virus, a subtype I-AB strain, was isolated from a donkey brain in Trinidad and was passaged 13 times in embryonated eggs. The virus was attenuated by 78 passages in fetal guinea pig heart (FGPH) cell cultures, plaque-purified in chick embryo fibroblasts (CEFs), and passaged four additional times in FGPH cell cultures. The VEE TC-83 virus designation is a direct reference to the 83 passages in cell culture. VEE C-84 vaccine is formalin-inactivated and is made from the TC-83 production seed, which was TC-82 that had undergone one additional passage in CEFs. This C-84 production seed is passaged once more in CEFs to derive the C-84 vaccine, which is inactivated with 0.1% formalin and then freeze-dried. The inactivation procedure is based on that used by Salk and colleagues to inactivate the poliovirus. TC-83 and C-84 contain streptomycin and neomycin, each at a concentration of 50 µg/mL. Laboratory infections with epizootic VEEV strains closely related to the parent strain have essentially been eliminated since the introduction of these vaccines and improvements in personal protective equipment.

Immunologically naïve people at risk for exposure to VEE received a single dose of the live-attenuated TC-83 vaccine. Those who seroconvert (PRNT ₈₀ titer ≥1:20) receive a single booster of C-84 as needed based on their titer (PRNT ₈₀ <1:20); nonresponders to TC-83 receive a booster with C-84. The response rate to TC-83 alone was 82%. When TC-83 was followed by a single boost with C-84, a combined response rate of well over 90% was observed. A subsequent study found that over 95% of TC-83 nonresponders seroconverted after a three-dose series of C-84 (unpublished research). Female responders to TC-83 tended to have titers similar to those of male responders, but the frequency of nonresponders tended to be higher among women than men; in one study, the initial response rate among recipients of a single dose of TC-83 was 74% and 85%, for women and men respectively. The nature of this sex difference is not understood.

Approximately 23% of persons receiving the live-attenuated TC-83 sustain adverse reactions, including headache, sore throat, malaise, fatigue, myalgias, arthralgias, chills, and fever, symptoms similar to those seen following natural VEEV infection but less severe. The local reaction rate was less than 5%. The inactivated vaccine C-84 had a local reaction rate of approximately 5%, but essentially no systemic reactions were associated with its administration. Diabetes mellitus, abortion, and teratogenesis have been epidemiologically associated with natural wild-type VEEV infection and animal challenge studies have shown similar findings. Before pregnancy testing prior to vaccination became available, three cases of spontaneous abortion or stillbirth were temporally related to the administration of TC-83. However, VEEV was not recovered from culture of tissues in either case. (Though they were reported to FDA, these cases were never published as case reports.) With the advent of pregnancy testing, great care was taken to ensure that women were not pregnant before administration of TC-83. Out of an abundance of caution, persons with a family history of diabetes mellitus were considered ineligible for vaccination with TC-83, despite the lack of evidence for a causal association between diabetes mellitus and VEEV infection or vaccination with TC-83.

The ideal VEE vaccine would have a high seroconversion rate (>95%) and a low reaction rate (<5%). By these standards, TC-83 is reactogenic and has a moderate response rate, as measured by neutralizing antibody. In addition, TC-83 does not protect adequately against distantly related VEEV subtype I-AB variants or the other enzootic VEEV subtypes II through VI. Finally, the manufacturing process for TC-83 required the manipulation of infectious viral particles in a biosafety level 3 containment laboratory.

A live-attenuated vaccine candidate, V3526, used twin site-directed mutagenesis of the full-length complementary DNA clone of the virulent virus RNA. V3526 has two deletion mutations—a lethal deletion at the PE-2 cleavage signal site and a suppressor mutation at site 253 of the E1 glycoprotein—that should prevent reversion to wild-type VEEV. This vaccine candidate also has limited potential for transmission by mosquitoes and elicits cross-protection against different viral strains. After showing promise in preclinical studies, V3526 elicited the development of impressive neutralizing antibody levels in human volunteers during Phase I clinical trials. However, the vaccine was associated with a high frequency of fever and other flu-like symptoms; thus, further development was discontinued.

Because of the long history of frequent adverse reactions related to live-attenuated VEE vaccines, the manufacturer decided to inactivate V3526 and further develop it as an inactivated vaccine to be used as a priming vaccine. Testing showed reduced infectivity by both formalin inactivation (fV3526) and gamma-irradiation (gV3526). For example, both inactivated vaccine candidates showed a loss of neurovirulence after intracerebral inoculation of suckling BALB/c mice, suggesting that the vaccines were completely inactivated. Both fV3526 and gV3526 elicited robust immune responses; furthermore, protection was demonstrated by both vaccine candidates against subcutaneous challenge with VEEV I-AB Trinidad donkey strain in BALB/c mice following two doses of either vaccine. ^, Recently, researchers have been testing these vaccine candidates, using various adjuvants and routes of administration, against subcutaneous and aerosol challenge.

Other technologies are being evaluated for the production of vaccines against VEE as well. For example, Sharma and colleagues found that the hydrophobic alkylating compound 1,5-iodonaphthylazide can effectively inactivate virulent VEEV strain V3000. The resulting inactivated vaccine candidate was efficacious in protecting mice from virulent VEEV challenge, and adjuvants enhanced the efficacy. This methodology was also used to develop a candidate inactivated V3526 (INA-iV3526), which provided complete protection against an aerosol challenge with virulent VEEV in a mouse model. Rossi and colleagues ^, developed a live-attenuated vaccine using an encephalomyocarditis virus internal ribosome entry site (IRES)—a construct that inhibits translation of viral proteins in mosquito cells, thus preventing transmission by the natural VEEV vector. This IRES-based VEE vaccine fully protected mice and macaques from clinical disease after aerosol challenge with virulent VEEV.

DNA vaccines are also under development and, in protective efficacy studies in both mice and guinea pigs, have shown promise. One DNA vaccine candidate has also demonstrated protection against aerosol challenge with wild-type VEEV in nonhuman primates. ^, A Phase I clinical trial of the DNA vaccine candidate pWRG/VEE delivered as a single dose by intramuscular or intradermal electroporation showed the candidate to be safe, tolerable, and immunogenic in humans; with a dose-dependent VEEV-neutralizing antibody response, that was typically more robust than that seen with the intramuscular electroporation route.

A candidate based on a novel approach to DNA vaccine development, infectious DNA (iDNA), was protective in one BALB/c mouse challenge study. This technology was further used to develop a candidate (V4020) derived from TC-83, with genetically engineered attenuating mutations to prevent reversion. Preclinical research of V4020 in BALB/c mice and nonhuman primates has shown the candidate to be effective against challenge with wild-type VEEV TrD strain. First-in human studies of V4020 are planned in the near future.

Western Equine Encephalitis Virus

An inactivated WEE vaccine, TSI-GSD 210, was used at Fort Detrick since the 1970s to immunize at-risk laboratory personnel. The WEE vaccine is a lyophilized product derived from supernatant fluids of primary CEF cell cultures infected with the attenuated CM4884 strain of WEEV. ^, The supernatant fluid is harvested and filtered, the virus is inactivated with formalin, and the final product is lyophilized for storage at −20°C. The vaccine contains 50 µg/mL of neomycin. The primary end point used to measure immunogenicity of the WEE vaccine for biocontainment suite entry was a PRNT ₈₀ , titer of at least 1:40.

In an analysis of data from 363 volunteers who received 0.5 mL of inactivated TSI-GSD 210 vaccine subcutaneously at days 0, 7, and 28, 151 subjects (41.6%) responded with a PRNT ₈₀ titer of 1:40 or greater, whereas 212 subjects (58.4%) failed to achieve this neutralizing antibody titer. Of 115 initial nonresponders, 76 (66.1%) converted to responder status after a single booster. Kaplan–Meier plots showed that a regimen consisting of three initial doses and one booster induced a PRNT ₈₀ titer of at least 1:40 lasting 1.6 years in 50% of initial responders. Local and systemic adverse events were uncommon with this vaccine. Among 363 vaccinees receiving three initial injections of the WEE vaccine, only five reported local or systemic reactions (P.R. Pittman, P.H. Gibbs, and T.L. Cannon, unpublished data). A further report of the use of this vaccine in 1362 laboratory workers from 1987 to 2011 showed a PRNT ₈₀ titer at least 1:40 at day 56 in 40.7% of study participants after receipt of the three dose primary series. Overall, post booster dose there were 65.1% participants who demonstrated a PRNT ₈₀ titer at least 1:40. Approximately 24% of participants reported a systemic adverse event, and 76% reported a local adverse event. No instances of occupational WEE were documented among laboratory workers who develop neutralizing antibodies following vaccination.

A new lot of the WEE vaccine, Western Equine Encephalitis Vaccine, Inactivated, TSI-GSD 210, Lot 3-1-92, was found to be safe and immunogenic in a Phase I clinical trial. In this study, WEE vaccine was administered in 0.5-mL doses subcutaneously in the upper outer aspect of the triceps in a three-dose primary series (days 0, 7, and 28) with a mandatory boost (day 180). All 10 subjects were classified as responders at day 56. For 4 of 10 subjects, the titers had waned below the acceptable level by month 6. Following the month 6 boost, all 10 subjects developed titers above 1: 40, and all remained above this level for at least 1 year.

DNA-based vaccines against WEE have shown promise in challenge models in mice. One such study evaluated a DNA vaccine, pVHX-6, expressing the 26 S structural gene of WEEV strain 71 V-1658. All mice receiving four intraepidermal doses of pVHX-6 survived challenge with a homologous strain, but only 62% and 50% survived challenge with Fleming and CBA87 strains, respectively. Other studies have demonstrated, in a murine challenge model, the efficacy of DNA vaccines expressing the capsid and envelope proteins of WEEV. In two studies, a replication-defective human adenovirus serotype-5 (HAd5) was used as a vector for vaccine delivery. In the first study, a HAd5 vector encoding E2 and E1, administered as a single dose, conferred protection against challenge with homologous and heterologous WEEV strains. ^, In the second study, a HAd5 vector encoding E1 alone, designated adenovirus (Ad) serotype-5-E1 (Ad5-E1), provided total protection in mice against challenge with homologous and heterologous strains of WEEV. A potential drawback of this approach in humans is the widespread prevalence of antibodies against adenovirus.

Eastern Equine Encephalitis Virus

The EEE vaccine (TSI-GSD 104) is a lyophilized product originating in primary CEF cell cultures infected with the attenuated PE-6 strain of EEEV. The seed for the EEE vaccine was passaged twice in adult mice and twice in guinea pigs, then passaged nine times in embryonated eggs, followed by three passages in CEFs. The supernatant fluid, which was harvested and filtered, contained 50 µg each of neomycin and streptomycin and 0.25% w/v (weight per volume) of human serum albumin, USP (U.S. Pharmacopoeia). The virus was then inactivated with 0.05% formalin. When inactivation was completed, the residual formalin was neutralized by treatment with sodium bisulfite. The final product was lyophilized for storage at −20°C.

Among 255 volunteers who received the two-dose primary series of EEE vaccine (administered subcutaneously) between 1992 and 1998, 197 (77.3%) responded with a PRNT ₈₀ titer of 1: 40 or greater. Of initial nonresponders, 66% subsequently seroconverted following receipt of an EEE vaccine booster (administered intradermally). Among initial responders whose titers waned over time, 98.6% responded to a booster dose of EEE vaccine. Local and systemic side effects are infrequent, occurring in less than 1% of vaccinees after the primary vaccine series and in 3.7% after the first booster. Kaplan–Meier plots showed that two primary doses and one intradermal booster of TSI-GSD 104 provided satisfactory neutralizing anti-EEEV antibodies in 50% of initial responders for up to 2.2 years. Further study of this vaccine between 2002 and 2008 evaluated a three-dose primary series (days 0, 7, and 28 administered subcutaneously) and demonstrated a PRNT ₈₀ titer of at least 1:40 in 54% of participants. Booster doses of the vaccine were offered to those with PRNT ₈₀ titer less than 1:40. Participants were followed with annual titer draws for the duration of their participation in the study. During the life of the protocol the cumulative response rate (PRNT ₈₀ ≥1:40) of these annual titers was 59%. Between 2008 and 2016, a change was made to lengthen the vaccine dose intervals of the three-dose primary series (days 0 and 28 administered subcutaneously, month 6 administered intradermally), this change demonstrated a significantly superior immune response with a PRNT ₈₀ of at least 1:40 in 84% of participants. Booster doses of the vaccine were offered to those with PRNT ₈₀ titers <1:40. Participants were followed with annual titer draws for the duration of their participation in the study. During the life of the protocol the cumulative response rate (PRNT ₈₀ ≥1:40) of these annual titers was 75%. Between these two studies, systemic adverse events were associated with 5.9% of vaccine doses, and local adverse events were associated with 22.4% of vaccine doses. Among recent findings regarding next-generation EEE vaccines, a single subcutaneous dose of an EEE vaccine candidate, attenuated via an IRES, protected 100% of vaccinated mice against intraperitoneal challenge. An EEEV replicon and a combined VEEV/WEEV/EEEV replicon protected macaques from aerosol challenge. Preliminary work on development of a live-attenuated EEEV vaccine has resulted in the identification of multiple potential sites for attenuation, with some products having a combination of these attenuations resulting in 100% protection of vaccinated mice after both subcutaneous and intracerebral infection challenges.

Trivalent Equine Encephalitis Vaccines

Next-generation technologies have helped to develop several trivalent equine encephalitis vaccine candidates in recent years. The first uses the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform; a highly attenuated vaccinia virus adapted to chicken embryo fibroblasts, which has a well-described robust safety and immunogenicity profile and has the capacity to express numerous genes. This platform has been used to develop monovalent equine encephalitis vaccines (MVA-VEEV, MVA-WEEV, MVA-EEEV) and a trivalent equine encephalitis vaccine candidate (MVA-WEV). Use of these MVA-BN®-based vaccines in BALB/c mice has shown them to be safe and immunogenic, and displayed protective efficacy against homologous aerosol challenges in BALB/c mice. ^, The trivalent MVA-WEV candidate has completed a Phase I human dose escalation clinical trial (NCT04131595) of two doses of the vaccine given 4 weeks apart, with results currently pending. A trivalent DNA vaccine (3-EEV) has been shown to be immunogenic in mice and rabbits; and protective in mice against aerosol challenge. Another trivalent equine encephalitis vaccine candidate under development is composed of equine encephalitis virus-like particles (VLPs). A trivalent VLP vaccine candidate (WEVEE) was safe and elicited protective responses in both mice and nonhuman primates against aerosol challenges. Using passive transfer of IgG from immunized nonhuman primates to mice generated a protective effect, demonstrating a humoral mechanism of protection. A Phase I dose escalation study of a two-dose series of WEVEE given 8 weeks apart either alone or mixed with alum adjuvant has been completed (NCT03879603), with results currently pending.

Chikungunya Virus

CHIKV is spread primarily by Aedes aegypti and Aedes albopictus mosquitos and can cause an acute viral syndrome in humans characterized by symptoms ranging from mild to acute fever, including severe joint pain, muscle pain, headache, nausea, fatigue, rash, and arthritis. Persistent arthralgia is observed in a subset of patients (e.g., ranging from 14% to 68% depending on time and assessment), and is associated with female gender, older age, some comorbidities and the severity of the acute phase arthritis. Although documented epidemics have occurred since the late 18th century, the virus was first isolated during the 1952–1953 Tanzanian epidemic. During 2004–2006, an epidemic ravaged the Indian Ocean islands east of Madagascar—La Réunion, Mauritius–Seychelles, and Mayotte. More than 2 million people have been affected since the start of that epidemic, which has since spread to the Caribbean and India, with cases also occurring in persons from the United States, Europe, and elsewhere who traveled to the Caribbean. In December 2014, Kendrick and colleagues reported on the first 11 cases of CHIK originating in the United States (Florida). Genomic sequencing of six isolates from the Indian Ocean outbreak suggested that the strain responsible for the outbreak was related to East African isolates. In addition, evidence from the sequence data suggests that the CHIKV strain responsible for the Indian Ocean outbreak evolved, during the course of the outbreak, into several distinct variants. One mutation may have allowed the CHIKV to be efficiently transmitted by the most abundant mosquito species on La Réunion ( Aedes albopictus ); this may help explain the noted robustness of the epidemic. CHIKV is now widely distributed across the Southern hemisphere, with unpredictable regularity of epidemic waves, and was highlighted as a major public health risk by the WHO in 2017, and is also on the WHO neglected tropical disease priority list.

Following failed attempts to develop an effective inactivated vaccine during the 1960–1980s, a live-attenuated vaccine was made from the seed of the African green monkey (AGM) kidney vaccine, using CHIKV strain 15561, which had originally been isolated from an infected patient during a 1962 CHIK epidemic in Thailand. The resulting CHIKV 181/clone 25 vaccine, attenuated through multiple passages in AGM kidney cells followed by passage in Medical Research Council 5 (MRC-5) cells, was efficacious in challenge models in suckling mice and in nonhuman primates. A randomized, double-blind, placebo-controlled trial of the CHIK 181/clone 25 vaccine documented a seroconversion rate of 98% among alphavirus-naïve volunteers. One year after immunization, 85% of vaccinees remained seropositive. Injection site and systemic symptoms, including flu-like symptoms, were similar in vaccine and placebo recipients. However, the vaccine was temporally associated with arthralgia in 8% of vaccinees. One volunteer in the vaccine group developed a pruritic, eczema-like rash at the injection site. Interference between the CHIK 181/clone 25 vaccine and the live-attenuated VEE TC-83 vaccine is discussed below.

Additional strategies for the development of a safe and efficacious vaccine for CHIK are being pursued. These include chimeric alphavirus vaccine candidates using VEEV-attenuated vaccine strain TC-83, a naturally attenuated strain of EEEV, or Sindbis virus as a backbone with the structural protein genes of CHIKV. Wang and colleagues found that each of these chimeras produced robust neutralizing antibody responses, and vaccinated mice were fully protected against disease and viremia after CHIKV challenge. One DNA vaccine candidate expressing a component of the CHIKV envelope glycoprotein produced neutralizing antibodies in mice and macaques. A second DNA vaccine candidate used a plasmid coding for the CHIKV-capsid, E1 and E2. When injected into mice, this construct induced both broad cellular immunity and humoral responses to the recognized native antigen, detected by enzyme-linked immunosorbent assay (ELISA). Another group is evaluating a formalin-inactivated Vero cell–adapted vaccine candidate prepared using a strain from the India epidemic. Most recently, a promising virus-like particle (VLP) vaccine containing the viral envelope has elicited high titers of neutralizing antibodies in nonhuman primates and in human volunteers. In addition, a single immunization with a measles virus–vectored CHIK vaccine candidate induced high CHIKV antibody titers and protected mice from a lethal CHIKV challenge. This vaccine showed seroconversion rates ranging from 50.0% to 95.9% depending on the dose and administration schedule, and was well tolerated in a Phase II trial among healthy adults.

Finally, a live-attenuated vaccine based on La Reunion strain of East Central South African genotype has been developed by reverse genetics resulting in 60aa deletion within the C-terminal part of the nonstructural nsP3 gene of the viral replicase complex; reducing the in vivo viral replication capability and with no risk of reversion to wild-type. Challenge studies in NHPs with this candidate (VLA1553) demonstrated protection against viremia and disease.

Due to the travel vaccine market, commercial CHIKV vaccine development is in advanced stages. ^, However, the populations most affected by Chikungunya are in LMICs. CEPI therefore cofunded CHIKV vaccine development with the European Commission in 2018, and three candidates were granted funding: First, the live-attenuated virus vaccine candidate VLA1553 which in a recent Phase III trial in >4100 subjects achieved its primary endpoint by inducing protective CHIKV neutralizing antibody titers in 98.5% of subjects 28 days after one dose, and was well tolerated across all age groups. The program was awarded FDA Fast Track designation in 2018, European Medicines Agency (EMA)’s PRIME designation in 2020 and Breakthrough Therapy Designation by the FDA in 2021. The two other candidates funded by CEPI are the recombinant measles virus vaccine candidate MV-CHIK/V184 (Merck & Co., Inc., United States), which has completed Phase II and is preparing for Phase III trials, and an inactivated whole virion vaccine BBV87 (Bharat Biotec, India), which is entering Phase II/III trials in Latin America and Asia (ClinicalTrials.gov Identifier: NCT04566484). The unpredictable timing and locations of future Chikungunya outbreaks pose a critical challenge, as clinical efficacy evaluation in this setting requires tailored trial designs and rapid initiation of trial sites in outbreak locations. Building on the assumption that neutralizing antibodies are considered critical for protective immunity, an immunobridging approach to conditional authorization based on correlates of protection may accelerate development ahead of future outbreaks. This critical regulatory innovation was done for VLA1553 candidate in its pivotal Phase III trial, where a seroprotective titer was agreed upon with the FDA to serve as a surrogate of protection that can be utilized in a potential FDA submission under the accelerated approval pathway.

Finally, access to affordable and reliable diagnostics to estimate incidence pretrial and verify cases during efficacy trials is also a challenge with Zika and dengue being included in the differential diagnoses. The lack of a true insight in the disease burden also affects the impact on decisions to introduce future chikungunya vaccines.

Ross River Virus

Ross River Virus Disease (RRVD), or epidemic polyarthritis, was first recognized in Australia in 1928; Ross River virus (RRV), its causative agent, was first isolated in 1963 from a pool of mosquitoes. RRVD is essentially limited to Australia, Fiji, and the surrounding islands, including American Samoa and the Cook Islands, where several thousand cases occur per year. ^, In humans, polyarthritis may be followed by fever, rash, and lethargy; symptoms generally resolve after 3–6 months. In 1997, U.S. Marines participating in Operation Tandem Thrust in Queensland, Australia, had an infection rate of 1.5%; RRVD developed in nine individuals.

A vaccine candidate was derived from a virus isolated from a human case of classical RRVD using the C6-36 cell line ( A. albopictus ). The candidate underwent four serial passages in MRC-5 human fetal lung cells followed by two passages in Vero cells. Cell cultures contained penicillin (100 U/mL) and streptomycin (100 µg/mL). The virus was inactivated using binary ethyleneimine. In mice, the vaccine induced neutralizing antibodies and conferred protection against viremia after intravenous challenge with live RRV.

More recently, a Vero cell–grown, formalin-inactivated, benzonase treated (to digest host cell DNA), sucrose gradient–purified vaccine candidate induced neutralizing antibodies in mice. Immunized mice and guinea pigs failed to develop viremia following intravenous challenge with the prototype strain of RRV (T48). In a Phase I/II dose escalation study in 382 healthy, RRV-naïve adults, the vaccine was safe and immunogenic when administered as three immunizations (at days 0 and 21 and month 6) at four-dose levels, with or without aluminum hydroxide added as an adjuvant. The adjuvanted 2.5-µg dose stimulated the highest immune response. This group recently completed a Phase III study in 1755 healthy younger adults (ages 16–59 years) and 209 healthy older adults (ages 60 and older). The vaccine was well tolerated. Neutralizing antibody titers (≥1 : 10) were achieved by 91.5% of the younger adults and 76.0% of the older adults after the third vaccination. The potential for antibody-dependent enhancement of disease, which has been described for RRV in vitro, will need to be considered during the development of vaccines against RRV.

Alphavirus Interference

A recent study using replicon technology in both mice and macaques showed that individual replicon vaccine candidates for VEE and EEE or a combined VEEV/WEEV/EEEV replicon particle vaccine elicited strong neutralizing antibodies and protection against aerosol challenge with VEEV subtype I-AB (Trinidad donkey strain) and EEEV. However, the individual WEEV replicon and the combined VEEV/WEEV/EEEV replicon vaccine elicited low levels of neutralizing antibodies to WEEV and conferred poor protection in macaques. In particular, Wolfe and colleagues argue that an FDA-licensed trivalent VEE/WEE/EEE vaccine—one that overcomes the safety, immunogenicity, and immune interference issues of the existing IND vaccines—is required to meet the requirements of the biodefense community. Such a vaccine also would be of great value to the public health and agricultural communities.

Experience with sequential administration of alphavirus vaccines at USAMRIID led to several interesting observations relating to immunologic interference. Prior immunization with inactivated EEE and/or WEE vaccines decreases one’s ability to mount a neutralizing antibody response following receipt of live-attenuated VEE TC-83 vaccine. However, no interference is observed when the order of administration is reversed, when VEE TC-83 vaccine is given first. Similarly, interference occurs when two live-attenuated alphavirus vaccines, VEE TC-83 and a CHIK vaccine (CHIK 181/clone 25), are administered sequentially. Volunteers initially vaccinated with VEE TC-83 exhibited poor neutralizing antibody responses to live-attenuated CHIK vaccine (46% response rate). Among persons initially inoculated with the CHIK vaccine or placebo and then given live-attenuated VEE TC-83 vaccine, geometric mean antibody titers to VEEV, as measured by 80% plaque reduction neutralization test (PRNT ₈₀ ), were uniformly depressed in previous CHIK vaccine recipients compared with placebo recipients. Interference may be an important consideration for the development of next-generation alphavirus vaccines, and particularly for the development of multiagent alphavirus vaccines.

Rift Valley Fever Virus

RVFV is a negative sense single-stranded RNA virus of the Bunyaviridae order and the Phlebovirus genus, first identified in 1931 during a farm outbreak among sheep in Kenya. The three-segmented genome of ~12 kb encodes a viral RNA polymerase, a precursor protein cleaved to form envelope glycoproteins G1 and G2, two nonstructural proteins, the nucleocapsid protein N and the major virulence factor nonstructural protein (NSs). RVFV is mosquito borne and its main animal hosts are sheep and other ruminants (e.g., cattle, goats, deer, gazelles, and antelopes). Humans are infected through mosquito bites, contact with fluids or parts of infected animals by respiratory or pharyngeal acquisition, and potentially milk. In humans, the incubation period is 2–6 days, and disease most often occurs in individuals with a history of recent contact with sheep or cattle. It can present with mild illness such as headache, myalgia, arthralgia and rash with rapid recovery, or in 8–10% of cases progress to more severe disease with eye lesions, encephalitis, and neurological sequelae, or further on to hemorrhagic fever (<1% of patients) with jaundice and other signs of liver impairment, followed by vomiting blood, bloody stool, or other bleeding 2–4 days after onset of illness. The case fatality rate (CFR) for hemorrhagic fever cases is around 50%. In animals, RVFV causes severe disease with fever, weakness, and loss of pregnancy (up to 100% abortion rate), and most young animals will die.

Under appropriate climatologic conditions, explosive epizootics among animals and epidemics in humans occur with considerable morbidity and mortality.

Over the period of 1931–2020, RVFV spread throughout Africa and into the Arabian Peninsula and some countries in western Asia. Increased seroprevalence in both humans and animals over this period suggests potentially increased RVFV exposure, with sheep most likely exposed during RVF outbreaks. Outbreaks in humans have ranged in size from a few cases to thousands of cases, with case fatality risk ranging from 1% to 30%. Over the course of 45 years a total of 10,923 human RVF cases were reported; most from Sudan, Egypt, and Kenya although an estimated 20,000–100,000 people may have been infected in South Africa in the 1950s. The consequences of further spread into naïve animal and human populations would be potentially devastating. RVFV largely affects people living in pastoral communities in low-income and middle-income countries, and outbreaks can therefore have potential devastation in terms of large-scale agricultural economies and cause substantial social disruption. The development of a vaccine or other countermeasures against the RVF virus is important for the protection of human and animal populations in Africa and the Middle East, expatriate personnel involved in those regions, and livestock and people in the United States and elsewhere.

The limited antigenic diversity and a single serotype presents an opportunity for RVFV vaccine development. Multiple preclinical RVFV vaccines have been developed and two live-attenuated vaccines have been evaluated in humans; however none are licensed for human use. There is no correlate of protection for RVF defined, but studies in animals have shown that neutralizing antibodies, mainly targeting the surface glycoproteins (Gn/Gc), are sufficient to protect from virulent RVFV challenge. Mouse studies have shown that CD4+ and CD8+ T cells may be involved in modulating RVFV disease and that IFN-α responses contribute to protection. In humans very few studies of natural immunity have been performed, however immunization with a formalin-inactivated RVFV vaccine conferred long-lived, RVFV-specific T-cell responses for up to 24 years.

The U.S. Army developed two vaccines to combat this threat: an inactivated RVF vaccine (TSI-GSD 200) and, more recently, a live-attenuated product (RVF MP-12; TSI-GSD 223). The Entebbe strain of RVF, isolated from a mosquito pool in Bwamba County, Uganda, is the source for the inactivated vaccine. The virus was passaged 184 times in adult mice, followed by two passages in fetal rhesus lung (FRhL) cells to form the production seed. Although the original vaccine was produced in primary African green monkey kidney cells, the current vaccine lots are produced in FRhL cells. ^, The vaccine was inactivated in 0.05% formalin. Following verification of viral inactivation, the residual formaldehyde was neutralized with sodium bisulfite to less than 0.01%. A study of the immunogenicity and safety of inactivated RVF vaccine in humans during a 12-year period showed that the vaccine was safe and immunogenic when the three-dose primary series and one booster were administered. ^, In one study, 540 (90%) of 598 volunteers given three 1.0-mL doses of TSI-GSD 200 subcutaneously on days 0, 7, and 28 responded with a PRNT ₈₀ titer of 1:40 or greater. Three-fourths of the initial nonresponders developed a PRNT ₈₀ titer of 1:40 or greater after a single booster. However, approximately 10% of recipients of the inactivated RVF vaccine required repeated boosting. In these individuals, a booster typically resulted in an adequate titer, which waned over the next year to <1:40, prompting another booster (P.R. Pittman, unpublished data).

An isolated RVF strain recovered from a nonfatal human case that occurred during the first Egyptian epidemic in 1977 was used to derive the live-attenuated RVF MP-12 vaccine. The virus (ZH548) was passaged twice in suckling mouse brain, then once in FRhL cells. It was then attenuated by 12 serial alternating passages in human lung cell cultures (MRC-5 cells, certified for vaccine use) by previously described methods in the presence or absence of 5-fluorouracil. The resulting RVF MP-12 vaccine is a lyophilized product originating from supernatant fluids harvested from the final mutagenesis passage. The vaccine has undergone extensive safety testing and challenge studies in several animal species: rodents, sheep (including pregnant ewes and naïve neonatal lambs), cattle (including in utero–vaccinated bovids), and monkeys. Furthermore, Miller and colleagues showed that RVF MP-12 vaccine, administered to sheep, resulted in long-lasting immunity and has limited potential to be transmitted to mosquitoes feeding on vaccinated animals.

RVF MP-12 has undergone clinical evaluation in human volunteers at USAMRIID. In a Phase I randomized, double-blind, dose-escalation/route-seeking study, 56 healthy, nonpregnant subjects were randomly selected to receive RVF MP-12 (10 ^4.7 plaque-forming units [PFU] subcutaneously, n = 10; 10 ^3.4 PFU intramuscularly, n = 6; or 10 ^4.4 PFU intramuscularly, n = 27) or placebo ( n = 13). Only infrequent and minor side effects were seen among placebo and MP-12 recipients. One volunteer had a titer of 1.3 log by direct plaquing in cell culture. Six vaccinees had transient low-titer viremia detected by amplification only; all six of these volunteers were from the group receiving 10 ^4.4 PFU intramuscularly. Neutralizing antibodies (measured by PRNT ₈₀ titer), as well as RVF-specific immunoglobulin Ig M and IgG, were observed in 40 (93%) of 43 vaccine recipients. The highest peak geometric mean antibody titers were observed in the group receiving 10 ^4.4 PFU intramuscularly. Overall, 28 (82%) of 34 RVF MP-12 recipients available for testing remained seropositive (PRNT ₈₀ ≥ 1:20) at 1 year following inoculation. A joint program between the University of Texas Medical Branch and USAMRIID for further development of the RVF MP-12, funded by the National Institute of Allergy and Infectious Diseases, involved the administration of a single intramuscular dose of RVF MP-12 vaccine to 19 naïve male and nonpregnant female subjects. The vaccine was safe and immunogenic in this vaccine trial. A total of 18 (95%) of 19 subjects developed neutralizing antibodies against RVF virus, as determined by a PRNT ₈₀ titer of 1:20 or more. Results suggested that the vaccine resulted in, at most, only low-level viremia. No virus was detected by direct plaque assay; however, during the first 14 days after vaccination, nine MP-12 isolates were recovered from five subjects with the use of amplification by blind, double passage in Vero cells. No single-nucleotide polymorphisms or reversions were observed in the attenuating mutations of the parent virus.

Another RVF live-attenuated vaccine candidate is Clone 13. This vaccine candidate is a plaque-purified clone of RVF virus that contains a large deletion in the small (S) genome segment that disrupts the biological functions of the nonstructural proteins (NSs). Clone 13 has proven highly immunogenic in mice, sheep, and goats, although it has demonstrated only moderate immunogenicity in cattle. ^, Other approaches to the development of vaccines against RVF virus, all of which are at the preclinical stage, include vaccines based on viral vectors ; DNA vaccines with molecular adjuvants ; subunit vaccines based on purified proteins ; and vaccines based on single-cycle replicable vaccine mutants.

The 2019 WHO Target Product Profile (TPP) for RVFV vaccines called for human vaccines for reactive use in outbreak settings with rapid onset of immunity and for long-term protection of persons at high ongoing risk ( https://www.who.int/news-room/articles-detail/rift-valley-fever-vaccines-target-product-profile ). Single-dose regimens were preferred, with suitability for a wide population including pregnant women if feasible. Guided by this, CEPI decided to fund two live-attenuated vaccine RVFV candidates (RVFV-4s and DDVax) that both are in preclinical development, with the aim of continuing into phase 1 studies to assess the safety, tolerability, and immunogenicity of a single-dose approach. The RVFV-4s candidate from the Wageningen Bioveterinary Research, Netherlands is a four-segmented RVFV variant vaccine developed by splitting the M-genome segment into two M-type segments each encoding one of the structural glycoproteins (Gn or Gc). RVFV-4s was shown to confer protective immunity after a single dose, with absence of teratogenic effects in pregnant ewes. The second DDVax candidate is developed by Colorado State University, United States and has removed key genes for virulence, also stopping the virus from replicating in mosquitoes (recombinant RVF ZH501 strain encoding deletions of both NSs and NSm genes). This vaccine candidate was protective in pregnant ewes at 42 days of pregnancy, with no detectable adverse effects in newborn lambs.

Key challenges for developing human vaccines for RVF disease are the limited understanding of human immunity to RVF and the lack of epidemiologic studies with appropriate diagnostics to inform efficacy evaluation and intervention strategies. Particular attention is paid to ensure safety in pregnant women, assessing the risk of reassortment between live, attenuated RVF vaccines, and wild-type strains and what constitutes an acceptable benefit:risk ratio. Introduction of a preventive RVF vaccine for humans may be challenging due to a low and unpredictable disease burden, however better surveillance data and mathematical modeling can guide the best use of vaccines in a one health perspective to reduce the risk posed by RVF to human health.