Flow virometry for process monitoring of live virus vaccines-lessons learned from ERVEBO.

Direct at line monitoring of live virus particles in commercial manufacturing of vaccines is challenging due to their small size. Detection of malformed or damaged virions with reduced potency is rate-limited by release potency assays with long turnaround times. Thus, preempting batch failures caused by out of specification potency results is almost impossible. Much needed are in-process tools that can monitor and detect compromised viral particles in live-virus vaccines (LVVs) manufacturing based on changes in their biophysical properties to provide timely measures to rectify process stresses leading to such damage. Using ERVEBO, MSD's Ebola virus vaccine as an example, here we describe a flow virometry assay that can quickly detect damaged virus particles and provide mechanistic insight into process parameters contributing to the damage. Furthermore, we describe a 24-h high throughput infectivity assay that can be used to correlate damaged particles directly to loss in viral infectivity (potency) in-process. Collectively, we provide a set of innovative tools to enable rapid process development, process monitoring, and control strategy implementation in large scale LVV manufacturing.

Effect of change in cell substrate on the critical quality attributes of L-Zagreb Mumps vaccine manufactured using parallel plate bioreactor.

Leningrad-Zagreb strain of mumps vaccine virus was grown on two different cell substrates viz. MRC-5 cells and Vero cells besides its original cell substrate i.e. Chicken Embryo Cells. Homogeneous virus pools prepared from each set of experiments were then lyophilized as per standard in-house protocol. Critical Quality Attributes (CQAs) such as the titer of the bulk vaccine and potency and stability of the lyophilized vaccine were then estimated using the CCID50 method to understand the lyophilization losses and thermal losses respectively in the vaccine. Another CQA viz. the genetic homogeneity of the vaccine was also tested using the single base extension method for identifying the nucleotides present at the three known locations of single nucleotide polymorphism (SNP). Comparison of CQA results across different cell substrates indicated encouraging results for Vero cell grown L-Zagreb virus compared to the MRC-5 cells grown L-Zagreb mumps virus. Significant improvement in productivity was also observed in the dynamic culture conditions compared to the static culture conditions. Progressive work in this research area can lead to development of a cGMP manufacturing process for mumps vaccine with easy scale up potential in future.

Avian leukosis virus contamination in live vaccines: A retrospective investigation in China.

Avian leukosis (AL) is one of the most pandemic immunosuppressive diseases and has been widely spread between 2006 and 2009 in China. The contamination of avian leukosis virus (ALV) in attenuated vaccine is considered as one of the possible transmission routes of this disease. Based on a retrospective survey of 918 batches of attenuated vaccine produced before 2010, three of them were identified as ALV-positive and corresponding ALV strains were successfully isolated from a live Fowlpox virus vaccine, a live Newcastle disease virus vaccine and a live Infectious Bursal Disease virus vaccine, respectively, and whole-genome sequencing showed that these three isolates shared the highest homology with ALV-A wild strains isolated in China (97.7%) over the same period, and the phylogenetic analysis based on their gp85 genes further confirmed that they belong to subgroup A. Meanwhile, although these three ALV-A strains isolated from contaminated vaccines shared a close genetic relationship, their U3 region of genome have a relatively low identity, suggesting that these three strains may have different sources. This study reminds us once again that the possibility of ALV infecting chickens through contaminated live vaccines, requiring us to carry out stricter exogenous virus monitoring in vaccines.

The Use of Next-Generation Sequencing for the Quality Control of Live-Attenuated Polio Vaccines.

BACKGROUND: Next-generation sequencing (NGS) analysis was compared to the current MAPREC (mutational analysis by polymerase chain reaction and restriction enzyme cleavage) assay for quality control of live-attenuated oral polio vaccine (OPV). METHODS: MAPREC measures reversion of the main OPV attenuating mutations such as uracil (U) to cytosine (C) at nucleotide 472 in the 5' noncoding region of type 3 OPV. Eleven type 3 OPV samples were analyzed by 8 laboratories using their in-house NGS method. RESULTS: Intraassay, intralaboratory, and interlaboratory variability of NGS 472-C estimates across samples and laboratories were very low, leading to excellent agreement between laboratories. A high degree of correlation between %472-C results by MAPREC and NGS was observed in all laboratories (Pearson correlation coefficient r = 0.996). NGS estimates of sequences at nucleotide 2493 with known polymorphism among type 3 OPV lots also produced low assay variability and excellent between-laboratory agreement. CONCLUSIONS: The high consistency of NGS data demonstrates that NGS analysis can be used as high-resolution test alternative to MAPREC, producing whole-genome profiles to evaluate OPV production consistency, possibly eliminating the need for tests in animals. This would be very beneficial for the quality assessment of next-generation polio vaccines and, eventually, for other live-attenuated viral vaccines.

The Quest for a Truly Universal Influenza Vaccine.

There is an unmet public health need for a universal influenza vaccine (UIV) to provide broad and durable protection from influenza virus infections. The identification of broadly protective antibodies and cross-reactive T cells directed to influenza viral targets present a promising prospect for the development of a UIV. Multiple targets for cross-protection have been identified in the stalk and head of hemagglutinin (HA) to develop a UIV. Recently, neuraminidase (NA) has received significant attention as a critical component for increasing the breadth of protection. The HA stalk-based approaches have shown promising results of broader protection in animal studies, and their feasibility in humans are being evaluated in clinical trials. Mucosal immune responses and cross-reactive T cell immunity across influenza A and B viruses intrinsic to live attenuated influenza vaccine (LAIV) have emerged as essential features to be incorporated into a UIV. Complementing the weakness of the stand-alone approaches, prime-boost vaccination combining HA stalk, and LAIV is under clinical evaluation, with the aim to increase the efficacy and broaden the spectrum of protection. Preexisting immunity in humans established by prior exposure to influenza viruses may affect the hierarchy and magnitude of immune responses elicited by an influenza vaccine, limiting the interpretation of preclinical data based on naive animals, necessitating human challenge studies. A consensus is yet to be achieved on the spectrum of protection, efficacy, target population, and duration of protection to define a "universal" vaccine. This review discusses the recent advancements in the development of UIVs, rationales behind cross-protection and vaccine designs, and challenges faced in obtaining balanced protection potency, a wide spectrum of protection, and safety relevant to UIVs.

Pharmacovigilance capacity strengthening for WHO prequalification: The case of the trivalent influenza vaccine manufactured by Instituto Butantan.

Instituto Butantan is a biomedical research center and vaccine manufacturer affiliated with the São Paulo State Secretary of Health in Brazil. In 2013, Instituto Butantan successfully licensed its trivalent influenza vaccine, in order to support the Brazilian National Immunization Program's influenza vaccination strategy, which was introduced in 1999. In order to respond to the increasing influenza vaccine demand worldwide, Instituto Butantan is undergoing prequalification of its trivalent influenza vaccine by the World Health Organization (WHO). A key requirement of the prequalification review was the submission of a pharmacovigilance plan, including an active surveillance evaluation, for the trivalent influenza vaccine, and proof of a functional pharmacovigilance system at Instituto Butantan. The aim of this paper is to describe the capacity strengthening process of the pharmacovigilance system at Instituto Butantan for the WHO prequalification of the trivalent influenza vaccine. This process was supported by PATH and the U.S. Federal Government Biomedical Advanced Research and Development Authority (BARDA). The key strategic axes for this capacity strengthening process included the improvement of organizational structure, human resources training, internal processes and procedures, appropriate documentation, and acquisition of an E2B compliant pharmacovigilance database. The project led to the establishment of a functional pharmacovigilance system compliant with international regulatory requirements.

Efficacy of Type 2 PRRSV vaccine against challenge with the Chinese lineage 1 (NADC30-like) PRRSVs in pigs.

The objective of the present study was to determine the cross-protection of Ingelvac PRRS MLV against challenge with the new lineage 1 PRRSV emerged in China in pigs. Two lineage 1 PRRSV strains (FJZ03 and FJWQ16 originated from recombination event between NADC30 and JXA1-like strain). We found that pigs vaccinated with the vaccine were protected against challenge with the FJZ03 as shown by fewer days of clinical fever, reduced lung pathology scores, lower PRRS virus load in the blood and developed broadly neutralizing antibodies with high titers to FJZ03. In contrast, vaccine provided limited protection against challenge with FJWQ16 with higher fever, lower antibody titers, lower neutralizing antibodies and higher viral loads in blood. These results demonstrate PRRSV-MLV provides incomplete protection against new lineage 1 PRRSVs.

Shedding of Salmonella Typhimurium in vaccinated and unvaccinated hens during early lay in field conditions: a randomised controlled trial.

BACKGROUND: Salmonella vaccination is one of the control measure that farmers can use to reduce bacterial shedding in their flocks. This study aimed to examine the efficacy of the Vaxsafe® ST (Strain STM-1) attenuated live vaccine administered as ocular and oral doses followed by an intramuscular (IM) dose in rearing, in reducing contamination by Salmonellae of both eggs and the environment in the commercial multi-age cage layer sheds. A randomised controlled trial was conducted up to 26 weeks post last vaccine on two different multi-age caged egg farms. RESULTS: No clinical symptoms were observed following IM administration of STM-1 during rearing. Following the first two STM-1 doses, both vaccinated and unvaccinated birds exhibited antibody titres below the positive cut-off value, however after IM administration of STM-1, antibody titres in the vaccinated group were above the cut-off value. Wild type Salmonella Typhimurium was not detected during the rearing of pullets. During production, the antibody titres were significantly higher in the vaccinated group at all sampling points during this trial. There was no significant difference in the prevalence of Salmonella (detected by culture and PCR method) between the vaccinated and unvaccinated groups on the egg belt and faeces in early lay. Wild-type Salmonella spp. were consistently found in dust samples. Quantitative PCR (qPCR) assay was able to differentiate between the live vaccine strain and wild type Salmonella. The load of wild-type Salmonella in shed environment was relatively low (1.3 log10 ± 0.48 CFU/m2 of surface area). CONCLUSION: Given that Salmonella Typhimurium and other serovars are able to survive/persist in the shed environment (such as in dust), regular cleaning and or removal of dust from shed is important. Use of the Vaxsafe® ST vaccine in multi-age flocks is "not an ultimate intervention" for reduction of Salmonella Typhimurium because of the complexities involved in achieving control, such as the efficacy of cleaning of sheds, the lack of resting periods between batches and the possible carry over of contamination from existing flocks. Hence implementation of more than one or several interventions strategies is essential.

Incidence of live-attenuated influenza vaccine administration beyond expiry date in children and adolescents aged 2-17 years in the UK: a population-based cohort study.

OBJECTIVES: To estimate the proportion of live-attenuated influenza vaccine (LAIV) doses administered beyond expiry date in children and adolescents during influenza seasons 2013-2014 and 2014-2015 in the UK. DESIGN: This was a retrospective cohort study. Two cohorts of children and adolescents who received LAIV from 1 September 2013 to 31 March 2014 and from 1 September 2014 to 31 March 2015 and aged 2-17 years at time of LAIV administration were identified from the Clinical Practice Research Datalink (CPRD). SETTING: More than 500 primary care practices in the UK. POPULATION: Proportions of vaccine doses administered beyond expiry date were assessed among 47 396 and 67 099 LAIV recipients with a documented vaccine lot identifier in influenza seasons 2013-2014 and 2014-2015, respectively. INTERVENTION: None. MAIN OUTCOME MEASURE: Administrations of expired LAIV were ascertained by comparison of vaccination dates in CPRD records with expiration dates in AstraZeneca/MedImmune lot distribution data. RESULTS: Overall, 245 LAIV recipients, 80 in 2013-2014 and 165 in 2014-2015, received a dose after its expiration date, yielding proportion estimates of 1.7 per 1000 doses (95% CI 1.3 to 2.1) in season 2013-2014 and 2.5 per 1000 (95% CI 2.1 to 2.8) in season 2014-2015. This proportion increased above 1.0% after December during each season. Most (84% in influenza season 2013-2014 and 59% in influenza season 2014-2015) received an expired dose <30 days after its expiration date. The proportion was higher in London (relative risk 1.93 (95% CI 1.25 to 2.99)) and when the number of LAIV recipients registered in the practice was lower than the median number per practice (relative risk 2.69 (95% CI 1.99 to 3.62)). CONCLUSIONS: Administration of expired LAIV doses occurs infrequently.

Safety of measles-containing vaccines in post-marketing surveillance in Anhui, China.

The safety of measles vaccination is of great interest and importance to public health practice and the general society. We have analyzed the adverse events following immunization (AEFIs) of currently used measles-containing vaccines (including live attenuated measles vaccine, live attenuated measles and rubella combined vaccine, live attenuated measles and mumps combined vaccine, live attenuated Measles, Mumps and Rubella Combined Vaccine) in Anhui Province, China. From 2009 to 2014, 9.9 million doses of measles-containing vaccines were administrated and 1893 AEFIs were found (191.4 per million doses), of which, 33 serious AEFIs (3.3 per million vaccine doses) were reported. 59.4% (1124 cases) were male cases, and 85.1% (1611 cases) occurred in persons aged < 1 year. 93.3% (1766 cases) occurred at the first dose of vaccination and 95.9% (1815 cases) were found within 3 days after vaccination. This study presents up-to-date data and suggests that the measles-containing vaccines used in Anhui Province of China are safe.

Time for Genome Editing: Next-Generation Attenuated Malaria Parasites.

Immunization with malaria parasites that developmentally arrest in or immediately after the liver stage is the only way currently known to confer sterilizing immunity in both humans and rodent models. There are various ways to attenuate parasite development resulting in different timings of arrest, which has a significant impact on vaccination efficiency. To understand what most impacts vaccination efficiency, newly developed gain-of-function methods can now be used to generate a wide array of differently attenuated parasites. The combination of multiple attenuation approaches offers the potential to engineer efficiently attenuated Plasmodium parasites and learn about their fascinating biology at the same time. Here we discuss recent studies and the potential of targeted parasite manipulation using genome editing to develop live attenuated malaria vaccines.

Development of Vaccines for Chikungunya Fever.

Chikungunya fever, an acute and often chronic arthralgic disease caused by the mosquito-borne chikungunya virus (CHIKV), has reemerged since 2004 to cause millions of cases. Because CHIKV exhibits limited antigenic diversity and is not known to be capable of reinfection, a vaccine could serve to both prevent disease and diminish human amplification during epidemic circulation. Here, we review the many promising vaccine platforms and candidates developed for CHIKV since the 1970s, including several in late preclinical or clinical development. We discuss the advantages and limitations of each, as well as the commercial and regulatory challenges to bringing a vaccine to market.

The Long-Term Safety, Public Health Impact, and Cost-Effectiveness of Routine Vaccination with a Recombinant, Live-Attenuated Dengue Vaccine (Dengvaxia): A Model Comparison Study.

BACKGROUND: Large Phase III trials across Asia and Latin America have recently demonstrated the efficacy of a recombinant, live-attenuated dengue vaccine (Dengvaxia) over the first 25 mo following vaccination. Subsequent data collected in the longer-term follow-up phase, however, have raised concerns about a potential increase in hospitalization risk of subsequent dengue infections, in particular among young, dengue-naïve vaccinees. We here report predictions from eight independent modelling groups on the long-term safety, public health impact, and cost-effectiveness of routine vaccination with Dengvaxia in a range of transmission settings, as characterised by seroprevalence levels among 9-y-olds (SP9). These predictions were conducted for the World Health Organization to inform their recommendations on optimal use of this vaccine. METHODS AND FINDINGS: The models adopted, with small variations, a parsimonious vaccine mode of action that was able to reproduce quantitative features of the observed trial data. The adopted mode of action assumed that vaccination, similarly to natural infection, induces transient, heterologous protection and, further, establishes a long-lasting immunogenic memory, which determines disease severity of subsequent infections. The default vaccination policy considered was routine vaccination of 9-y-old children in a three-dose schedule at 80% coverage. The outcomes examined were the impact of vaccination on infections, symptomatic dengue, hospitalised dengue, deaths, and cost-effectiveness over a 30-y postvaccination period. Case definitions were chosen in accordance with the Phase III trials. All models predicted that in settings with moderate to high dengue endemicity (SP9 ≥ 50%), the default vaccination policy would reduce the burden of dengue disease for the population by 6%-25% (all simulations: -3%-34%) and in high-transmission settings (SP9 ≥ 70%) by 13%-25% (all simulations: 10%- 34%). These endemicity levels are representative of the participating sites in both Phase III trials. In contrast, in settings with low transmission intensity (SP9 ≤ 30%), the models predicted that vaccination could lead to a substantial increase in hospitalisation because of dengue. Modelling reduced vaccine coverage or the addition of catch-up campaigns showed that the impact of vaccination scaled approximately linearly with the number of people vaccinated. In assessing the optimal age of vaccination, we found that targeting older children could increase the net benefit of vaccination in settings with moderate transmission intensity (SP9 = 50%). Overall, vaccination was predicted to be potentially cost-effective in most endemic settings if priced competitively. The results are based on the assumption that the vaccine acts similarly to natural infection. This assumption is consistent with the available trial results but cannot be directly validated in the absence of additional data. Furthermore, uncertainties remain regarding the level of protection provided against disease versus infection and the rate at which vaccine-induced protection declines. CONCLUSIONS: Dengvaxia has the potential to reduce the burden of dengue disease in areas of moderate to high dengue endemicity. However, the potential risks of vaccination in areas with limited exposure to dengue as well as the local costs and benefits of routine vaccination are important considerations for the inclusion of Dengvaxia into existing immunisation programmes. These results were important inputs into WHO global policy for use of this licensed dengue vaccine.

Development and approval of live attenuated influenza vaccines based on Russian master donor viruses: Process challenges and success stories.

Influenza is a viral infection that affects much of the global population each year. Vaccination remains the most effective tool for preventing the disease. Live attenuated influenza vaccine (LAIV) has been used since the 1950s to protect humans against seasonal influenza. LAIVs developed by the Institute of Experimental Medicine (IEM), Saint Petersburg, Russia, have been successfully used in Russia since 1987. In 2006, the World Health Organization (WHO) announced a Global action plan for influenza vaccines (GAP). WHO, recognizing potential advantages of LAIV over the inactivated influenza vaccine in a pandemic situation, included LAIV in the GAP. BioDiem Ltd., a vaccine development company based in Melbourne, Australia which held the rights for the Russian LAIV, licensed this technology to WHO in 2009. WHO was permitted to grant sub-licenses to vaccine manufacturers in newly industrialized and developing countries to use the Russian LAIV for the development, manufacture, use and sale of pandemic and seasonal LAIVs. To date, WHO has granted sub-licenses to vaccine manufacturers in China (Changchun BCHT Biotechnology Co., Ltd.), India (Serum Institute of India Pvt. Ltd.) and Thailand (Government Pharmaceutical Organization). In parallel, in 2009, IEM signed an agreement with WHO, under which IEM committed to supply pandemic and seasonal candidate vaccine viruses to the sub-licensees. This paper describes the progress made by collaborators from China, India, Russia and Thailand in developing preventive measures, including LAIV against pandemic influenza.

Safety and immunogenecity of a live attenuated Rift Valley fever vaccine (CL13T) in camels.

BACKGROUND: Rift Valley fever is an emerging zoonotic viral disease, enzootic and endemic in Africa and the Arabian Peninsula, which poses a significant threat to both human and animal health. The disease is most severe in ruminants causing abortions in pregnant animals, especially sheep animals and high mortality in young populations. High mortality rates and severe clinical manifestation have also been reported among camel populations in Africa, to attend however none of the currently available live vaccines against RVF have been tested for safety and efficacy in this species. In this study, the safety and efficacy (through a neutralizing antibody response) of the thermostable live attenuated RVF CL13T vaccine were evaluated in camels in two different preliminary experiments involving 16 camels, (that 12 camels and 4 pregnant camels). RESULTS: The study revealed that the CL13T vaccine was safe to use in camels and no abortions or teratogenic effects were observed. The single dose of the vaccine stimulated a strong and long-lasting neutralizing antibody response for up to 12 months. CONCLUSION: The presence of neutralization antibodies is likely to correlate with protection; however protection would need to be confirmed by challenge experiments using the virulent RVF virus.

Generation and protective efficacy of a cold-adapted attenuated avian H9N2 influenza vaccine.

To prevent H9N2 avian influenza virus infection in chickens, a long-term vaccination program using inactivated vaccines has been implemented in China. However, the protective efficacy of inactivated vaccines against antigenic drift variants is limited, and H9N2 influenza virus continues to circulate in vaccinated chicken flocks in China. Therefore, developing a cross-reactive vaccine to control the impact of H9N2 influenza in the poultry industry remains a high priority. In the present study, we developed a live cold-adapted H9N2 influenza vaccine candidate (SD/01/10-ca) by serial passages in embryonated eggs at successively lower temperatures. A total of 13 amino acid mutations occurred during the cold-adaptation of this H9N2 virus. The candidate was safe in chickens and induced robust hemagglutination-inhibition antibody responses and influenza virus-specific CD4(+) and CD8(+) T cell immune responses in chickens immunized intranasally. Importantly, the candidate could confer protection of chickens from homologous and heterogenous H9N2 viruses. These results demonstrated that the cold-adapted attenuated H9N2 virus would be selected as a vaccine to control the infection of prevalent H9N2 influenza viruses in chickens.

Serotype, antigenicity, and pathogenicity of a naturally recombinant TW I genotype infectious bronchitis coronavirus in China.

Since 2009, strains of the naturally recombinant TW I genotype of infectious bronchitis virus (IBV) have caused considerable damage to the Chinese poultry industry. To better understand the antigenicity and pathogenesis of this genotype, the characteristics of the ck/CH/LDL/140520 strain were compared to those of four commercial IB vaccine strains that are used commonly in China, as well as four attenuated viruses that represent two types of IBV strains, which are believed to have originated in China and are the predominant IBV types circulating in chicken flocks in China and many other parts of the world. The results showed that all eight strains were genetically and serotypically different from the strain ck/CH/LDL/140520. Furthermore, neither the vaccine strains nor the attenuated viruses could provide complete respiratory protection of chickens against a challenge with the ck/CH/LDL/140520 strain, indicating that it is necessary to develop new live vaccines or to evaluate the use of established vaccines in combination to control naturally recombinant TW I-type IBV strains in the future. Our results showed that strain ck/CH/LDL/140520 is very pathogenic, and that it is able to cause cystic oviducts in a high percentage of birds, as well as mortality due to nephritis and respiratory distress with complete tracheal ciliostasis, especially in chickens infected at 1day of age.

Comparison of the humoral response between sheep vaccinated with a killed-virus vaccine and those vaccinated with a modified-live virus vaccine against bluetongue virus serotype 17.

OBJECTIVE To compare the humoral response between sheep vaccinated with a killed-virus (KV) vaccine and those vaccinated with a modified-live virus (MLV) vaccine against bluetongue virus (BTV) serotype 17. DESIGN Randomized clinical trial followed by a field trial. ANIMALS 30 yearling crossbred ewes (phase 1) and 344 sheep from 7 Wyoming farms (phase 2). PROCEDURES In phase 1, ewes seronegative for anti-BTV antibodies received sterile diluent (control group; n = 10) or an MLV (10) or KV (10) vaccine against BTV-17 on day 0. Ewes in the KV group received a second dose of the vaccine on day 21. Ewes were bred 5 months after vaccination and allowed to lamb. Anti-BTV antibodies were measured in ewes at predetermined times after vaccination and in their lambs once at 5 to 10 days after birth. In phase 2, 248 commercial sheep were screened for anti-BTV antibodies and vaccinated with a KV vaccine against BTV-17 on day 0. Sheep seronegative for anti-BTV antibodies on day 0 (n = 90) underwent follow-up serologic testing on day 365 along with 96 unvaccinated cohorts (controls). RESULTS In phase 1, all vaccinated ewes developed anti-BTV antibodies by 14 days after vaccination and remained seropositive for 1 year; all of their lambs were also seropositive. All control ewes and lambs were seronegative. In phase 2, the prevalence of vaccinated sheep with anti-BTV antibodies 1 year after vaccination was 93% and 76% as determined by a serum neutralization assay and competitive ELISA, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Both vaccines induced antibodies against BTV-17 that persisted for at least 1 year and provided passive immunity for lambs and may be a viable option to protect sheep against disease.

Long-Term Safety and Immunogenicity of a Tetravalent Live-Attenuated Dengue Vaccine and Evaluation of a Booster Dose Administered to Healthy Thai Children.

We evaluated the safety and immunogenicity of two doses of a live-attenuated, tetravalent dengue virus vaccine (F17/Pre formulation) and a booster dose in a dengue endemic setting in two studies. Seven children (7- to 8-year-olds) were followed for 1 year after dose 2 and then given a booster dose (F17/Pre formulation), and followed for four more years (Child study). In the Infant study, 49 2-year-olds, vaccinated as infants, were followed for approximately 3.5 years after dose 2 and then given a booster dose (F17) and followed for one additional year. Two clinically notable events were observed, both in dengue vaccine recipients in the Infant study: 1 case of dengue approximately 2.7 years after dose 2 and 1 case of suspected dengue after booster vaccinations. The booster vaccinations had a favorable safety profile in terms of reactogenicity and adverse events reported during the 1-month follow-up periods. No vaccine-related serious adverse events were reported during the studies. Neutralizing antibodies against dengue viruses 1-4 waned during the 1-3 years before boosting, which elicited a short-lived booster response but did not provide a long-lived, multivalent antibody response in most subjects. Overall, this candidate vaccine did not elicit a durable humoral immune response.