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Mosquito Control. Activity and Surveillance. Vaccinated mice demonstrated a low level of immunity, with neutralizing titers in the range of at four weeks following vaccination. However, the horse vaccine was later recalled in after reports of increased adverse events in horses following vaccination [ ]. A number of other live, attenuated recombinant virus vector vaccines have also been developed. A veterinary vaccine using a canarypox-based vector expressing WNV prM and Env has demonstrated efficacy in preventing viremia across several animal species including horses, cats and dogs [ 77 , 78 ].
An HIV-based lentiviral vector expressing the WNV Env protein was shown to induce a protective immune response in mice within 1 week of a single immunization [ 79 ].
A non-integrative version of this same vector system was later developed in an attempt to reduce safety concerns, and was also shown to induce protective immune responses in mice [ 80 ]. A WNV candidate based on an attenuated strain of the measles virus Schwarz strain expressing WNV Env was tested in both mice [ 81 ] and squirrel monkeys [ 82 ], with protection demonstrated against death or viremia, respectively.
While several of these approaches show promising results with regard to immunogenicity and protective efficacy in animal models, some practical constraints may limit their clinical utility. For vaccine approaches using lentiviral vectors [ 79 , 80 ] or VSV [ 83 ], can safety and biocontainment issues be adequately addressed? For candidates using adenovirus [ 84 ] and measles virus vectors [ 81 , 82 ], will pre-existing immunity in humans limit their use?
These questions may need to be addressed if these vaccine platforms are to move forward into clinical development. A number of preclinical studies have been reported using attenuated strains of WNV [ 85 - 90 ], created either through classic cell culture methods or targeted genetic mutations. Using this approach, investigators were able to induce neutralizing antibodies and protective immune responses in mice, hamsters [ 91 ] and NHP [ 92 ]. Results such as these hold promise as an additional avenue for development of a WNV vaccine, but live viral vaccines have been somewhat unpredictable in the past and may pose potential regulatory concerns for the elderly and immunocompromised, representing two vulnerable populations with the greatest need for a safe and effective WNV vaccine.
In terms of protein vaccines, three main types have been developed including chemically-inactivated whole virus, virus-like particle VLP , and recombinant WNV envelope subunit formulations Table 2.
The first successful veterinary vaccine, licensed in , was a formaldehyde-inactivated preparation of WNV-NY99, shown to protect against WNV challenge [ 93 ]. Though this vaccine was not developed for human use, it has provided an important proof-of-principle for this class of non-replicating protein vaccines against WNV disease. Vaccination elicited neutralizing antibody titers in mice, and although responses were relatively low average; they could be boosted with a monophosporyl lipid A MPL, a detoxified form of LPS and saponin-based liposomal adjuvant average; [ 94 ].
Several groups have developed vaccine candidates based on the DIII of the WNV Env protein, since this region of the Env has been shown to harbor potent neutralizing antibody epitopes in mice [ 95 ]. A recent report exploring the subunit WNV Env vaccine approach has provided further insight into the role of different adjuvants in this vaccine model system [ ]. Inactivated whole virus vaccines represent another important class of vaccine candidates Table 2.
The first licensed veterinary vaccine was based on this approach, using a formalin-inactivated crude viral harvest of WNV-NY99, formulated with a squalene-based adjuvant, to induce protective immunity in horses [ 93 ] as well as other animal models [ 72 ]. A formalin-inactivated vaccine based on a pathogenic lineage 1 strain of WNV ISR98 has also been described, and was shown to be protective in a goose challenge model [ ].
Two other groups have developed formalin-inactivated vaccines, both based on the virulent WNV-NY99 strain of virus [ , ].
From a clinical perspective, one concern for these formalin-inactivated WNV vaccine candidates is that they are based on pathogenic strains of WNV. The use of highly pathogenic strains of virus for inactivated vaccines creates logistical issues associated with the handling of BSL3 pathogens during large-scale cGMP manufacturing, in addition to safety concerns if complete inactivation is not achieved. For instance, one of the worst vaccine-related tragedies in the United States came from the improper inactivation of virulent poliovirus during vaccine manufacturing in i.
This resulted in , doses of vaccine that contained live poliovirus and resulted in 40, children who were infected, 56 who developed paralytic poliomyelitis, and 5 children died [ - ].
While modern manufacturing practices ensure the safety of the inactivated polio vaccine IPV , there has still been a push to further increase the safety margin of IPV by switching from current virulent poliovirus strains to attenuated virus vaccine strains [ ].
As an alternative to traditional formaldehyde-based vaccines, a novel hydrogen peroxide H 2 O 2 inactivation approach has been developed to produce a first-generation whole-virus vaccine against WNV [ 5 , ]. Since the introduction of WNV into the United States in , significant research efforts have been expended to create a viable vaccine for disease prevention in humans. To date, there have been eight published clinical trials assessing safety and immunogenicity across multiple vaccine platforms Table 3.
The trial was performed as an open-label Phase I study in healthy subjects aged years old. The vaccine was administered at 4 mg per dose intramuscularly using a needle-free injection system on days 0, 28, and Fifteen vaccinees were enrolled, with a total of 12 vaccinees completing the entire 3-dose regimen.
The most common side effects were limited to local injection site reactions, with no reports of serious adverse events. At week 12 approximately 1 month following the final immunization PRNT 50 titers ranged from - , with a group geometric mean of An alternative WNV reporter-virus particle RVP neutralization assay was also utilized to assess immunogenicity, with RVP neutralization titers ranging from —, The reason for the differences between the two assays is uncertain, though studies using WNV-specific monoclonal antibodies MAb indicate that the maturation state of the virus used in the RVP assay may play a role [ ].
In other vaccine models, international serum standards have proven useful for bridging the results obtained from different research groups [ ] and it may be worthwhile to standardize WNV neutralizing assays with a defined target virus e. In an effort to improve immunogenicity, a modified DNA plasmid construct incorporating an additional regulatory element from the human T cell leukemia virus type 1 HTLV-1 , in conjunction with the previously used CMV promoter, was tested in a Phase I clinical trial [ ].
The clinical protocol closely matched the previous study [ ] in terms of vaccination dose and booster regimen, but included both a young ages; and older ages; cohort, with 15 subjects enrolled per group.
As with the prior DNA construct, side effects were mild and generally limited to the site of injection. Neutralizing antibody titers against RVP indicated a trend toward higher antibody responses with the modified vector, although this was not statistically significant. However, PRNT 50 serum titers against WNV were not directly assessed in this second clinical trial, limiting the ability to make comparisons to other clinical studies.
Further development of the WNV DNA vaccine platform is unclear, with the most recent human clinical trial completed in , and the results published in [ ]. In general, DNA vaccines have suffered from concerns over immunogenicity and potential safety issues such as DNA integration into the genome [ ].
However, in this instance the WNV DNA vaccine was able to induce a measurable WNV-specific immune response and was well-tolerated without any serious adverse events [ ]. Improvements in DNA delivery technologies are continuing to occur [ ] and these innovations, combined with this promising vaccine candidate, may offer a path towards further development of a successful WNV-specific DNA vaccine.
To date, two live, attenuated chimeric flavivirus vaccine candidates have been tested in humans [ 70 , - ]. Viremia, as measured by the area under the curve AUC , was significantly higher with the lower dose vs. These titers fell to , and , by day Using a similar vaccine approach, others have indicated that differences in the target virus used in the neutralization assays may have a substantial impact on the outcome of PRNT titers when assessing clinical samples [ ].
In addition, prior studies in NHP vaccinated with chimeric DENV or JEV constructs demonstrated that PRNT 50 titers ranged from about 2- to fold lower when using wild type strains of target virus as compared to the homologous chimeric vaccine construct [ , ]. Following the Phase I results, two Phase II studies were performed using a plaque purified derivative of ChimeriVax-WN02, which had been developed in an effort to increase attenuation and reduce viremia in vaccinated subjects [ , ].
In part 1, a total of subjects were enrolled, receiving 3. In part 2, 96 subjects were enrolled and received only the 3. As with the Phase I trial, the vaccine was generally well tolerated across dosages and age groups.
Peak viremia was reduced in comparison to the Phase I study, though lower doses of virus still resulted in higher levels of viremia. In part 2 of the study, increased viremia was shown to be associated with advanced age. Although there were no severe adverse events observed in this small study, this result indicates that close monitoring of viremia should be continued in the future since the elderly represent the primary target population for a successful WNV vaccine.
Immunogenicity as judged by neutralization against the homologous vaccine virus increased with virus dose in part 1 and group PRNT 50 titers reached , by day 28 after vaccination with the highest dose of virus 3. In part 2 3. Subjects in part 2 were followed for up to 1 year, at which point neutralizing titers had declined to an average of PRNT 50 titers at day 28 were not statistically different between vaccine groups and averaged between , somewhat lower than that observed in elderly subjects in the prior trial [ ].
Additional immunogenicity time points were not assessed. Why viremia levels increased in this clinical trial compared to the prior Phase II study is unclear, though the demographics of the study population were more narrowly focused on older individuals. Further development of this WNV vaccine is uncertain since this program was suspended by Sanofi Pasteur after acquisition of Acambis in [ ].
Two cGMP lots were produced, with the second lot modified to contain additional non-coding point mutations in an effort to increase virus production from cell culture, though the amino acid sequence of the polyprotein did not differ between lots [ ]. The vaccine was found to be safe and well tolerated, with no statistically significant differences in injection site or systemic adverse events between vaccine and placebo groups. This continued the trend of an inverse relationship between virus dose and subsequent viremia levels previously established with the YFVD-based ChimeriVax platform [ ].
Group geometric mean PRNT 60 titers also varied with dose. At the 10 3 dose level, PRNT 60 titers peaked at day 42 with an average of range; , , while the 10 4 dose demonstrated a peak of range; , on day This vaccine was developed by Hawaii Biotech, and is based on a truncated WNV Env protein formulated with aluminum hydroxide.
Preclinical studies in multiple species including mice, birds and NHP, demonstrated the induction of WNV-specific neutralizing responses after vaccination [ , ]. In a Phase I clinical trial, a total of 24 subjects were enrolled to assess immunogenicity and safety [ ].
Immunizations were performed intramuscularly at weeks 0, 4 and 8 for a total of 3 inoculations per subject. The vaccine was well tolerated, with most side effects limited to injection site reactions. After the third immunization, mean titers in these two groups increased but appeared to remain within the range of The Phase I clinical trial of this WNV vaccine ended in but further clinical development appears to have stalled. While WNV-specific immunogenicity seems to be generally low, the use of advanced adjuvant systems such as saponin derivatives [ ] may improve vaccine potency and are worth exploring in future studies.
Before a vaccine can be licensed for commercial use, it must be shown to be both safe and effective at reducing the disease that it is designed to prevent. One of the biggest hurdles in reaching licensure for a human WNV vaccine is the limited feasibility to perform field efficacy trials.
Although WNV is endemic throughout the continental United States, the relatively low incidence and sporadic nature of WNV outbreaks poses challenges for study design and implementation.
One complicating factor is that even in these endemic locations, WNV disease activity varies greatly from year to year. However, if a systematic effort is put forth to test the efficacy of an advanced WNV vaccine, then it may be possible to vaccinate an at-risk population and monitor WNV disease activity over the course of seasons or until enough WNV cases have accumulated to provide statistical significance between vaccine and placebo groups.
By performing an efficacy trial at more than one location with historically high WNV incidence, the risk of failing to identify a statistically significant decrease in disease incidence may be further mitigated. Albeit logistically challenging, this long-term approach to disease monitoring during a WNV Phase III field trial may provide the feasibility necessary to determine vaccine efficacy and move an effective vaccine closer to licensure.
B Distribution of Americans aged 65 years and older based on the Census. Reported WNV cases declined during , but a substantial increase was observed in and , indicating that relatively high incident, recurrent WNV may be likely to continue in certain at-risk geographic areas.
As an alternative to field efficacy trials, some have suggested that the FDA Animal Rule may provide a route for licensure [ ]. However, the Animal Rule appears to have been primarily implemented in the context of bioterrorism threats wherein no other practical alternative exists [ ].
Since initial publication of the Animal Rule in , two drug products for the treatment of nerve gas poisoning have been approved following this mechanism, but no vaccines have advanced to licensure [ ].
As noted by the FDA, the Animal Rule does not provide a short-cut to licensure, and may actually take much longer than standard clinical testing [ ].
As another method to supporting innovations in medicine, the FDA has recently developed its Advancing Regulatory Science initiative, which has been suggested as an avenue for WNV vaccine development [ ], though how this would work in practice remains to be seen.
Outbreaks of WNV cause considerable morbidity, mortality and disease-associated economic loss. Although the economic impact of a successful vaccination program should not be the sole determinant involved in making public health decisions, it is nevertheless an important parameter in determining overall feasibility.
If the financial cost to a society is reduced through decreased disease burden, there will be more support for implementing a particular vaccine policy rather than when there is little or no cost advantage.
In , there were 4, reported cases of WNV in the U. Figure 1 , with cases identified in Louisiana [ ]. Albeit optimistic, if we assume that the earliest date that a vaccine could be commercially available is in 5 years e.
Even though the outbreaks in and were larger then that encountered in Figure 1 , over the past 10 years , the average annual number of reported cases of WNV in the U. In contrast to these rough estimates of cost, a formal analysis of the cost-effectiveness of WNV vaccination was performed in [ 31 ] and this study is frequently used as the basis for estimating the costs associated with more recent WNV outbreaks [ , ]. The Zohrabian et al.
One key parameter for determining economic cost is loss of productivity due to death or short-term and long-term disability [ 31 ]. However, since WNV disease disproportionally afflicts the aged population, there is consequently a lower base productivity profile and decreased lifespan potential.
The lack of a more humanitarian component to cost-effectiveness projections is a challenging issue, especially in cases in which cost-effectiveness is borderline or not cost-effective despite providing a mechanism to reduce clinical disease burden [ ].
Another key point with the Zohrabian study [ 31 ] is that it is based only on direct WNV-related health care costs and does not take into account the costs of WNV surveillance, vector control, and outbreak prevention and response costs, which can be large [ , ]. Further studies that incorporate these factors will be important for gauging the full economic impact of WNV outbreaks in the U. Instead of implementing mass vaccination, a more feasible and cost-effective approach to preventing WNV outbreaks in the U.
A targeted vaccine campaign could be designed based on vaccinating specific regions with the highest WNV disease incidence or the highest total number of reported WNV cases. Alternatively, the targeted vaccine campaign could be based on age, with the elderly representing the most at-risk population. WNV activity has been reported in all 48 contiguous states but the risk of contracting WNND varies substantially both between states and even within each state when monitored at the county level Figure 2A.
The low population density in the regions most greatly impacted by WNV provides further support for the approach of a targeted regional vaccine program instead of implementing mass vaccination of the nation at large, which will invariably include large populations at low historical risk for WNV disease.
Based on Census estimates Source: www. The total population of these states is million, making mass vaccination a potentially challenging prospect from a cost-effectiveness perspective. On the other hand, if focused only on the aged population, then a nation-wide vaccine plan could be implemented. In , approximately By using a targeted vaccine program aimed at the most at-risk populations across the nation at either the state or county level, a safe and effective WNV vaccine could sharply reduce disease burden and mortality while still providing substantial societal cost savings.
While several promising WNV vaccines have been evaluated in clinical trials over the last decade, a licensed human vaccine remains elusive. One concern for future vaccine development is the feasibility of performing Phase III efficacy trials for a zoonotic disease like WNV that is known for sporadic outbreaks that are often difficult to predict.
However, with detailed WNV epidemiologic data from across the US, it may be possible to identify locations for potential vaccine trials, especially in Midwestern regions with relatively low population density but high WNV disease incidence. Although universal WNV vaccination is unlikely to be cost-effective, further studies are needed to determine if targeted vaccine campaigns focused on at-risk age groups or geographical regions will provide a favorable cost:benefit ratio, especially in light of recent evidence indicating that almost 3 million Americans have likely been infected with WNV and most cases continue to go unreported.
The recognition of much larger WNV incidence, coupled with increasing evidence for long-term disability and decreased quality of life among WNV survivors, further indicates a compelling need for a safe and effective WNV vaccine. The next 5 years may be pivotal in terms of the continued successful development of a safe and effective vaccine against West Nile virus.
Several vaccine products are in various stages of clinical development and new vaccine technologies are still entering the pipeline. A targeted vaccine program has the potential to be cost-effective and there is renewed interest in WNV vaccine technology, especially since the outbreak of represented not only the largest outbreak since , but also resulted in the most WNV-associated deaths on record.
Despite the continued dedication to WNV-related research spanning the last 15 years, several key challenges remain. Due to the sporadic nature of WNV outbreaks and many mitigating factors e. Through the combined and collaborative efforts of WNV epidemiologists, virologists, vaccine manufacturers, state and county health officials, and regulatory agencies, it is possible that a safe and effective vaccine against West Nile virus can become a reality and provide protection to the vulnerable populations within our communities that need it most.
Based on more than a decade of surveillance in the United States, it is expected that WNV will continue to threaten vulnerable populations for the foreseeable future.
The severity of WNV disease is associated with advanced age and often results in long-term health issues including potentially severe neurological sequelae and a higher mortality rate after recovery from acute infection. More studies are needed to determine if WNV infection is linked to chronic kidney disease. Several early-stage vaccine clinical trials have been completed, but none have advanced to licensure.
Reference standards for performing WNV-specific neutralization assays, including highly characterized serum standards and reference strains of WNV, should be considered. A formal cost-benefit analysis of targeted WNV vaccination should be performed, preferably including not only direct health care costs but also costs associated with WNV surveillance, prevention, and outbreak response.
We thank Andrew Townsend for excellent graphical design and assistance. OHSU, Dr. Slifka, and Dr. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU.
No writing assistance was utilized in the production of this manuscript. National Center for Biotechnology Information , U. Plug-and-display: decoration of virus-like particles via isopeptide bonds for modular immunization.
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