Development of a next-generation chikungunya virus vaccine based on the HydroVax platform.

Chikungunya virus (CHIKV) is an emerging/re-emerging mosquito-borne pathogen responsible for explosive epidemics of febrile illness characterized by debilitating polyarthralgia and the risk of lethal infection among the most severe cases. Despite the public health risk posed by CHIKV, no vaccine is currently available. Using a site-directed hydrogen peroxide-based inactivation approach, we developed a new CHIKV vaccine, HydroVax-CHIKV. This vaccine technology was compared to other common virus inactivation approaches including ß-propiolactone (BPL), formaldehyde, heat, and ultraviolet (UV) irradiation. Heat, UV, and BPL were efficient at inactivating CHIKV-181/25 but caused substantial damage to neutralizing epitopes and failed to induce high-titer neutralizing antibodies in vaccinated mice. HydroVax-CHIKV and formaldehyde-inactivated CHIKV retained intact neutralizing epitopes similar to live virus controls but the HydroVax-CHIKV approach demonstrated a more rapid rate of virus inactivation. HydroVax-CHIKV vaccination induced high neutralizing responses to homologous and heterologous CHIKV clades as well as to other alphaviruses including Mayaro virus, O'nyong'nyong virus, and Una virus. Following heterologous infection with CHIKV-SL15649, HydroVax-CHIKV-immunized mice were protected against viremia, CHIKV-associated arthritic disease, and lethal CHIKV infection by an antibody-dependent mechanism. In contrast, animals vaccinated with Heat- or UV-inactivated virus showed no protection against viremia in addition to demonstrating significantly exacerbated CD4+ T cell-mediated footpad swelling after CHIKV infection. Together, these results demonstrate the risks associated with using suboptimal inactivation methods that fail to elicit protective neutralizing antibody responses and show that HydroVax-CHIKV represents a promising new vaccine candidate for prevention of CHIKV-associated disease.

Successful Vaccines.

Vaccines are considered one of the most important advances in modern medicine and have greatly improved our quality of life by reducing or eliminating many serious infectious diseases. Successful vaccines have been developed against many of the most common human pathogens, and this success has not been dependent upon any one specific class of vaccine since subunit vaccines, non-replicating whole-virus or whole-bacteria vaccines, and attenuated live vaccines have all been effective for particular vaccine targets. After completing the initial immunization series, one common aspect of successful vaccines is that they induce long-term protective immunity. In contrast, several partially successful vaccines appear to induce protection that is relatively short-lived and it is likely that long-term protective immunity will be critical for making effective vaccines against our most challenging diseases such as AIDS and malaria.

Heterogeneity and longevity of antibody memory to viruses and vaccines.

Determining the duration of protective immunity requires quantifying the magnitude and rate of loss of antibodies to different virus and vaccine antigens. A key complication is heterogeneity in both the magnitude and decay rate of responses of different individuals to a given vaccine, as well as of a given individual to different vaccines. We analyzed longitudinal data on antibody titers in 45 individuals to characterize the extent of this heterogeneity and used models to determine how it affected the longevity of protective immunity to measles, rubella, vaccinia, tetanus, and diphtheria. Our analysis showed that the magnitude of responses in different individuals varied between 12- and 200-fold (95% coverage) depending on the antigen. Heterogeneity in the magnitude and decay rate contribute comparably to variation in the longevity of protective immunity between different individuals. We found that some individuals have, on average, slightly longer-lasting memory than others-on average, they have higher antibody levels with slower decay rates. We identified different patterns for the loss of protective levels of antibodies to different vaccine and virus antigens. Specifically, we found that for the first 25 to 50 years, virtually all individuals have protective antibody titers against diphtheria and tetanus, respectively, but about 10% of the population subsequently lose protective immunity per decade. In contrast, at the outset, not all individuals had protective titers against measles, rubella, and vaccinia. However, these antibody titers wane much more slowly, with a loss of protective immunity in only 1% to 3% of the population per decade. Our results highlight the importance of long-term longitudinal studies for estimating the duration of protective immunity and suggest both how vaccines might be improved and how boosting schedules might be reevaluated.

Persistence of Neutralizing Antibody Responses Among Yellow Fever Virus 17D Vaccinees Living in a Nonendemic Setting.

BACKGROUND: The once-in-a-lifetime recommendation for vaccination against yellow fever virus (YFV) has been controversial, leading to increased scrutiny of the durability of immunity after 17D vaccination. METHODS: This is a cross-sectional analysis of 17D vaccinees living in nonendemic Portland, Oregon. Neutralization assays were used to determine YFV immunity. The relationships between 17D immunity and vaccination history, demographics, and travel were evaluated using nominal logistic regression. RESULTS: Seventy-one of 92 (77.2%) subjects were YFV seropositive (90 percent plaque reduction neutralization test ≥1:10) at all timepoints, and 24 of 38 (63.8%) were YFV seropositive at ≥10 years after single-dose vaccination. No relationship was found between YFV immunity and time in endemic countries, other flavivirus immunity, or demographics. Subjects were most likely to become seronegative between 3 and 12 years postvaccination (logistic regression, odds ratio [OR] = 1.75; 95% confidence interval [CI], 1.12-2.73). A comparison of our results and 4 previous studies of YFV nonendemic vaccinees found that overall, 79% (95% CI, 70%-86%) of vaccinees are likely to be seropositive ≥10 years postvaccination. CONCLUSIONS: These results suggest that 1 in 5 17D vaccinees will lack neutralizing antibodies at ~10 years postvaccination, and a booster vaccination should be considered for nonendemic vaccinees before travel to regions where there is a high risk of YFV transmission.

Durability of Vaccine-Induced Immunity Against Tetanus and Diphtheria Toxins: A Cross-sectional Analysis.

BACKGROUND: Many adult immunization schedules recommend that tetanus and diphtheria vaccination be performed every 10 years. In light of current epidemiological trends of disease incidence and rates of vaccine-associated adverse events, the 10-year revaccination schedule has come into question. METHODS: We performed cross-sectional analysis of serum antibody titers in 546 adult subjects stratified by age or sex. All serological results were converted to international units after calibration with international serum standards. RESULTS: Approximately 97% of the population was seropositive to tetanus and diphtheria as defined by a protective serum antibody titer of ≥0.01 IU/mL. Mean antibody titers were 3.6 and 0.35 IU/mL against tetanus and diphtheria, respectively. Antibody responses to tetanus declined with an estimated half-life of 14 years (95% confidence interval, 11-17 years), whereas antibody responses to diphtheria were more long-lived and declined with an estimated half-life of 27 years (18-51 years). Mathematical models combining antibody magnitude and duration predict that 95% of the population will remain protected against tetanus and diphtheria for ≥30 years without requiring further booster vaccination. CONCLUSIONS: These studies demonstrate that durable levels of protective antitoxin immunity exist in the majority of vaccinated individuals. Together, this suggests that it may no longer be necessary to administer booster vaccinations every 10 years and that the current adult vaccination schedule for tetanus and diphtheria should be revisited.

A hydrogen peroxide-inactivated virus vaccine elicits humoral and cellular immunity and protects against lethal West Nile virus infection in aged mice.

West Nile virus (WNV) is an emerging pathogen that is now the leading cause of mosquito-borne and epidemic encephalitis in the United States. In humans, a small percentage of infected individuals develop severe neuroinvasive disease, with the greatest relative risk being in the elderly and immunocompromised, two populations that are difficult to immunize effectively with vaccines. While inactivated and subunit-based veterinary vaccines against WNV exist, currently there is no vaccine or therapy available to prevent or treat human disease. Here, we describe the generation and preclinical efficacy of a hydrogen peroxide (H(2)O(2))-inactivated WNV Kunjin strain (WNV-KUNV) vaccine as a candidate for further development. Both young and aged mice vaccinated with H(2)O(2)-inactivated WNV-KUNV produced robust adaptive B and T cell immune responses and were protected against stringent and lethal intracranial challenge with a heterologous virulent North American WNV strain. Our studies suggest that the H(2)O(2)-inactivated WNV-KUNV vaccine is safe and immunogenic and may be suitable for protection against WNV infection in vulnerable populations.

Mechanisms that determine plasma cell lifespan and the duration of humoral immunity.

Humoral immunity following vaccination or infection is mainly derived from two types of cells: memory B cells and plasma cells. Memory B cells do not actively secrete antibody but instead maintain their immunoglobulin in the membrane-bound form that serves as the antigen-specific B-cell receptor. In contrast, plasma cells are terminally differentiated cells that no longer express surface-bound immunoglobulin but continuously secrete antibody without requiring further antigenic stimulation. Pre-existing serum or mucosal antibody elicited by plasma cells (or other intermediate antibody-secreting cells) represents the first line of defense against reinfection and is critical for protection against many microbial diseases. However, the mechanisms involved with maintaining long-term antibody production are not fully understood. Here, we examine several models of long-term humoral immunity and present a new model, described as the 'Imprinted Lifespan' model of plasma cell longevity. The foundation of this model is that plasma cells are imprinted with a predetermined lifespan based on the magnitude of B-cell signaling that occurs during the induction of an antigen-specific humoral immune response. This represents a testable hypothesis and may explain why some antigen-specific antibody responses fade over time whereas others are maintained essentially for life.

Campylobacter vaccination reduces diarrheal disease and infant growth stunting among rhesus macaques.

Campylobacter-associated enteric disease is estimated to be responsible for more than 160 million cases of gastroenteritis each year and is linked to growth stunting of infants living under conditions of poor sanitation and hygiene. Here, we examine naturally occurring Campylobacter-associated diarrhea among rhesus macaques as a model to determine if vaccination could reduce severe diarrheal disease and infant growth stunting. Compared to unvaccinated controls, there are no Campylobacter diarrhea-associated deaths observed among vaccinated infant macaques and all-cause diarrhea-associated infant mortality is decreased by 76% (P = 0.03). By 9 months of age, there is a 1.3 cm increase in dorsal length that equaled a significant 1.28 LAZ (Length-for-Age Z score) improvement in linear growth among vaccinated infants compared to their unvaccinated counterparts (P = 0.001). In this work, we show that Campylobacter vaccination not only reduces diarrheal disease but also potentially serves as an effective intervention that improves infant growth trajectories.

Active Circulation of Corynebacterium ulcerans among Nonhuman Primates.

Diphtheria is rare in the United States. and many industrialized nations due to development of an effective vaccine, coupled with high vaccination coverage. Although there is continued risk of importation and transmission of Corynebacterium diphtheriae, C. ulcerans has now become the dominant source of diphtheria cases among several European countries. Bearing this in mind, a better understanding of C. ulcerans biology is clearly needed. Here, we identified active transmission of toxigenic C. ulcerans among indoor- and outdoor-housed rhesus macaques based on diphtheria toxin-specific serology assays as well as direct isolation of C. ulcerans from a recently infected animal. In addition to animal-to-animal transmission, we found serological evidence indicative of potential human transmission. Together, these results provide new details on natural Corynebacterium transmission among nonhuman primates and emphasizes the importance of maintaining high vaccination coverage to reduce the risk of potential zoonotic infection. IMPORTANCE C. ulcerans represents an emerging zoonotic agent of diphtheria, but little is known about its transmission or maintenance among animal reservoirs. In these studies, we identified diphtheria outbreaks among both outdoor- and indoor-housed rhesus macaques and isolated a toxigenic strain of C. ulcerans from a recently infected animal. Retrospective analysis indicated that toxigenic Corynebacteria have been circulating among these primates for decades with the potential for rare zoonotic transmission to humans.

Reduced infant rhesus macaque growth rates due to environmental enteric dysfunction and association with histopathology in the large intestine.

Environmental enteric dysfunction is associated with malnutrition as well as infant growth stunting and has been classically defined by villous blunting, decreased crypt-to-villus ratio, and inflammation in the small intestine. Here, we characterized environmental enteric dysfunction among infant rhesus macaques that are naturally exposed to enteric pathogens commonly linked to human growth stunting. Remarkably, despite villous atrophy and histological abnormalities observed in the small intestine, poor growth trajectories and low serum tryptophan levels were correlated with increased histopathology in the large intestine. This work provides insight into the mechanisms underlying this disease and indicates that the large intestine may be an important target for therapeutic intervention.

Duration of humoral immunity to common viral and vaccine antigens.

BACKGROUND: Maintenance of long-term antibody responses is critical for protective immunity against many pathogens. However, the duration of humoral immunity and the role played by memory B cells remain poorly defined. METHODS: We performed a longitudinal analysis of antibody titers specific for viral antigens (vaccinia, measles, mumps, rubella, varicella-zoster virus, and Epstein-Barr virus) and nonreplicating antigens (tetanus and diphtheria) in 45 subjects for a period of up to 26 years. In addition, we measured antigen-specific memory B cells by means of limiting-dilution analysis, and we compared memory B-cell frequencies to their corresponding serum antibody levels. RESULTS: Antiviral antibody responses were remarkably stable, with half-lives ranging from an estimated 50 years for varicella-zoster virus to more than 200 years for other viruses such as measles and mumps. Antibody responses against tetanus and diphtheria antigens waned more quickly, with estimated half-lives of 11 years and 19 years, respectively. B-cell memory was long-lived, but there was no significant correlation between peripheral memory B-cell numbers and antibody levels for five of the eight antigens tested. CONCLUSIONS: These studies provide quantitative analysis of serologic memory for multiple antigens in subjects followed longitudinally over the course of more than one decade. In cases in which multiple exposures or repeated vaccinations were common, memory B-cell numbers did not correlate with antibody titers. This finding suggests that peripheral memory B cells and antibody-secreting plasma cells may represent independently regulated cell populations and may play different roles in the maintenance of protective immunity.

Vaccine-mediated protection against Campylobacter-associated enteric disease.

Campylobacter coli and Campylobacter jejuni are responsible for 400 million to 500 million cases of enteric disease each year and represent the most common cause of bacterial gastroenteritis worldwide. Despite its global importance, Campylobacter vaccine development has been hampered by the lack of animal models that recapitulate human disease pathogenesis. Here, we describe a naturally occurring Campylobacter-associated diarrhea model in outdoor-housed rhesus macaques. Using this model, we developed novel next-generation H2O2-based Campylobacter vaccines that induced strong antibacterial antibodies to multiple Campylobacter proteins including flagellin and provided up to 83% protection against severe C. coli-associated diarrhea. Whole-genome sequencing of circulating Campylobacter strains revealed little to no homology within lipooligosaccharide or capsular polysaccharide loci with the Campylobacter vaccine strains used in these studies, indicating that vaccine-mediated immunity was not restricted to a single homologous serotype. Together, these results demonstrate an important advance in vaccine development and a new approach to reducing Campylobacter-associated enteric disease.

Role of Multivalency and Antigenic Threshold in Generating Protective Antibody Responses.

Vaccines play a vital role in protecting our communities against infectious disease. Unfortunately, some vaccines provide only partial protection or in some cases vaccine-mediated immunity may wane rapidly, resulting in either increased susceptibility to that disease or a requirement for more booster vaccinations in order to maintain immunity above a protective level. The durability of antibody responses after infection or vaccination appears to be intrinsically determined by the structural biology of the antigen, with multivalent protein antigens often providing more long-lived immunity than monovalent antigens. This forms the basis for the Imprinted Lifespan model describing the differential survival of long-lived plasma cell populations. There are, however, exceptions to this rule with examples of highly attenuated live virus vaccines that are rapidly cleared and elicit only short-lived immunity despite the expression of multivalent surface epitopes. These exceptions have led to the concept that multivalency alone may not reliably determine the duration of protective humoral immune responses unless a minimum number of long-lived plasma cells are generated by reaching an appropriate antigenic threshold of B cell stimulation. Examples of long-term and in some cases, potentially lifelong antibody responses following immunization against human papilloma virus (HPV), Japanese encephalitis virus (JEV), Hepatitis B virus (HBV), and Hepatitis A virus (HAV) provide several lessons in understanding durable serological memory in human subjects. Moreover, studies involving influenza vaccination provide the unique opportunity to compare the durability of hemagglutinin (HA)-specific antibody titers mounted in response to antigenically repetitive whole virus (i.e., multivalent HA), or detergent-disrupted "split" virus, in comparison to the long-term immune responses induced by natural influenza infection. Here, we discuss the underlying mechanisms that may be associated with the induction of protective immunity by long-lived plasma cells and their importance in future vaccine design.

Advanced oxidation technology for the development of a next-generation inactivated West Nile virus vaccine.

West Nile virus (WNV) is the most frequent mosquito-borne disease reported in the continental United States and although an effective veterinary vaccine exists for horses, there is still no commercial vaccine approved for human use. We have previously tested a 3% hydrogen peroxide (H2O2)-based WNV inactivation approach termed, HydroVax, in Phase I clinical trials and the vaccine was found to be safe and modestly immunogenic. Here, we describe an advanced, next-generation oxidation approach (HydroVax-II) for the development of inactivated vaccines that utilizes reduced concentrations of H2O2 in combination with copper (cupric ions, Cu2+) complexed with the antiviral compound, methisazone (MZ). Further enhancement of this oxidative approach included the addition of a low percentage of formaldehyde, a cross-linking reagent with a different mechanism of action that, together with H2O2/Cu/MZ, provides a robust two-pronged approach to virus inactivation. Together, this new approach results in rapid virus inactivation while greatly improving the maintenance of WNV-specific neutralizing epitopes mapped across the three structural domains of the WNV envelope protein. In combination with more refined manufacturing techniques, this inactivation technology resulted in vaccine-mediated WNV-specific neutralizing antibody responses that were 130-fold higher than that observed using the first generation, H2O2-only vaccine approach and provided 100% protection against lethal WNV infection. This new approach to vaccine development represents an important area for future investigation with the potential not only for improving vaccines against WNV, but other clinically relevant viruses as well.

An observer blinded, randomized, placebo-controlled, phase I dose escalation trial to evaluate the safety and immunogenicity of an inactivated West Nile virus Vaccine, HydroVax-001, in healthy adults.

BACKGROUND: West Nile virus (WNV) is the most common mosquito-borne infection in the United States. HydroVax-001 WNV is a hydrogen peroxide inactivated, whole virion (WNV-Kunjin strain) vaccine adjuvanted with aluminum hydroxide. METHODS: We performed a phase 1, randomized, placebo-controlled, double-blind (within dosing group), dose escalation clinical trial of the HydroVax-001 WNV vaccine administered via intramuscular injection. This trial evaluated 1 mcg and 4 mcg dosages of HydroVax-001 WNV vaccine given intramuscularly on day 1 and day 29 in healthy adults. The two dosing groups of HydroVax-001 were enrolled sequentially and each group consisted of 20 individuals who received HydroVax-001 and 5 who received placebo. Safety was assessed at all study days (days 1, 2, 4 and 15 post dose 1, and days 1, 2, 4, 15, 29, 57, 180 and 365 post dose 2), and reactogenicity was assessed for 14 days after administration of each dose. Immunogenicity was measured by WNV-specific plaque reduction neutralization tests (PRNT50) in the presence or absence of added complement or by WNV-specific enzyme-linked immunosorbent assays (ELISA). RESULTS: HydroVax-001 was safe and well-tolerated as there were no serious adverse events or concerning safety signals. At the 1 mcg dose, HydroVax-001 was not immunogenic by PRNT50 but elicited up to 41% seroconversion by WNV-specific ELISA in the per-protocol population (PP) after the second dose. At the 4 mcg dose, HydroVax-001 elicited neutralizing antibody responses in 31% of the PP following the second dose. In the presence of added complement, PRNT50 seroconversion rates increased to 50%, and 75% seroconversion was observed by WNV-specific ELISA. CONCLUSIONS: The HydroVax-001 WNV vaccine was found to be modestly immunogenic and well-tolerated at all dose levels.

Protective immunity following vaccination: how is it defined?

Vaccination represents an important medical breakthrough pioneered by Edward Jenner over 200 years ago when he developed the world's first vaccine against smallpox. To this day, vaccination remains the most effective means available for combating infectious disease. There are currently over 20 vaccines licensed for use within the US with many more vaccines in the R&D pipeline. Although vaccines must demonstrate clinical efficacy in order to receive FDA approval, the correlates of immunity vary remarkably between different vaccines and may be based primarily on animal studies, clinical evidence, or a combination of these sources of information. Correlates of protection are critical for measuring vaccine efficacy but researchers should know the history and limitations of these values. As vaccine technologies advance, the way in which we measure and define protective correlates may need to evolve as well. Here, we describe the correlates of protective immunity for vaccines against smallpox, tetanus, yellow fever and measles and compare these to a more recently introduced vaccine against varicella zoster virus, wherein a strict correlate of immunity has yet to be fully defined.

Regulation of constitutive p50/c-Rel activity via proteasome inhibitor-resistant IkappaBalpha degradation in B cells.

Constitutive NF-kappaB activity has emerged as an important cell survival component of physiological and pathological processes, including B-cell development. In B cells, constitutive NF-kappaB activity includes p50/c-Rel and p52/RelB heterodimers, both of which are critical for proper B-cell development. We previously reported that WEHI-231 B cells maintain constitutive p50/c-Rel activity via selective degradation of IkappaBalpha that is mediated by a proteasome inhibitor-resistant, now termed PIR, pathway. Here, we examined the mechanisms of PIR degradation by comparing it to the canonical pathway that involves IkappaB kinase-dependent phosphorylation and beta-TrCP-dependent ubiquitylation of the N-terminal signal response domain of IkappaBalpha. We found a distinct consensus sequence within this domain of IkappaBalpha for PIR degradation. Chimeric analyses of IkappaBalpha and IkappaBbeta further revealed that the ankyrin repeats of IkappaBalpha, but not IkappaBbeta, contained information necessary for PIR degradation, thereby explaining IkappaBalpha selectivity for the PIR pathway. Moreover, we found that PIR degradation of IkappaBalpha and constitutive p50/c-Rel activity in primary murine B cells were maintained in a manner different from B-cell-activating-factor-dependent p52/RelB regulation. Thus, our findings suggest that nonconventional PIR degradation of IkappaBalpha may play a physiological role in the development of B cells in vivo.

Plasma cell survival in the absence of B cell memory.

Pre-existing serum antibodies play an important role in vaccine-mediated protection against infection but the underlying mechanisms of immune memory are unclear. Clinical studies indicate that antigen-specific antibody responses can be maintained for many years, leading to theories that reactivation/differentiation of memory B cells into plasma cells is required to sustain long-term antibody production. Here, we present a decade-long study in which we demonstrate site-specific survival of bone marrow-derived plasma cells and durable antibody responses to multiple virus and vaccine antigens in rhesus macaques for years after sustained memory B cell depletion. Moreover, BrdU+ cells with plasma cell morphology can be detected for 10 years after vaccination/BrdU administration, indicating that plasma cells may persist for a prolonged period of time in the absence of cell division. On the basis of these results, long-lived plasma cells represent a key cell population responsible for long-term antibody production and serological memory.

Pre-clinical development of a hydrogen peroxide-inactivated West Nile virus vaccine.

West Nile virus (WNV) is a mosquito-transmitted pathogen with a wide geographical range that can lead to long-term disability and death in some cases. Despite the public health risk posed by WNV, including an estimated 3 million infections in the United States alone, no vaccine is available for use in humans. Here, we present a scaled manufacturing approach for production of a hydrogen peroxide-inactivated whole virion WNV vaccine, termed HydroVax-001WNV. Vaccination resulted in robust virus-specific neutralizing antibody responses and protection against WNV-associated mortality in mice or viremia in rhesus macaques (RM). A GLP-compliant toxicology study performed in rats demonstrated an excellent safety profile with clinical findings limited to minor and transient irritation at the injection site. An in vitro relative potency (IVRP) assay was developed and shown to correlate with in vivo responses following forced degradation studies. Long-term in vivo potency comparisons between the intended storage condition (2-8°C) and a thermally stressed condition (40±2°C) demonstrated no loss in vaccine efficacy or protective immunity over a 6-month span of time. Together, the positive pre-clinical findings regarding immunogenicity, safety, and stability indicate that HydroVax-001WNV is a promising vaccine candidate.