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Because the noncytolytic process for virus clearance does not completely eliminate viral RNA from neurons, a mechanism for long-term immunologic control of virus replication is needed to prevent virus reactivation or progressive disease,. Antibody is likely to participate in control, as well as initial clearance. Maintaining adequate levels of antibody in the CNS for continued control of virus replication requires either passage of antibody from the blood into the brain parenchyma or local production by resident antibody-secreting cells. The BBB restricts the entry of proteins from the blood into the CNS, and although this function is compromised during the acute phase of infection, it is quickly repaired. Under normal conditions, levels of antibody in the brain are sustained at 1% of plasma levels that are likely to be inadequate for long-term prevention of virus reactivation. Therefore, resident long-lived antibody-secreting cells that can continue to produce antiviral antibody for a lifetime are a feature of recovery from most CNS virus infections. Long-term immune control of virus replication is not always successful, leading to recurrent or progressive neurologic disease–.
A number of vaccine development studies have been conducted using mainly the three most commonly applied alphavirus vectors, SFV, SIN and VEE. To demonstrate the variety of approaches available, replicon particles, naked RNA and layered DNA vectors have been employed. Each approach has commonly generated responses detected by cellular or humoral responses. In the case of vaccine development against lethal viruses (Table 1), non-viral infectious targets (Table 2) and tumors (Table 3) immunization has provided long-term protection against challenges with the disease-causing agents. Moreover, due to the pathogenicity of several alphaviruses, they are themselves credible targets for vaccine development. Therefore, a number of studies, particularly for VEE and CHIK (Table 4), have provided protection against challenges with virulent alphavirus strains.
The potential of alphaviruses causing global epidemics has placed additional concern on the needs of addressing biosafety issues. Generally, the strains used for vaccine development are attenuated. Furthermore, in the case using alphavirus particles, second generation helper vectors or split helper systems have been applied to prevent any production of wild-type-like replication-proficient particles through non-homologous recombination. Only recently alphavirus-based vaccines have been subjected to clinical trials. In this context, SFV vectors have been used for delivery of immunostimulatory genes. Furthermore, immunizations for the treatment of CMV and vaccinations against prostate and metastatic cancers have been conducted with alphavirus vectors and particles. The strength of applying alphaviruses is the generation of rapid transgene expression and the transient nature of expression. However, the full potential of alphavirus-based vaccines has not been explored yet. In the future, it is anticipated that additional, positive observations, particularly in the form of providing protection against challenges with lethal pathogens and tumors, will attract the enhanced application of alphaviruses for vaccine development.
Self-replicating RNA virus vectors have been subjected to several clinical studies, albeit at an inferior level in comparison to adenovirus, AAV and lentivirus vectors. For instance, healthy volunteers were subjected to low-dose (3 × 105 pfu) immunization with the VSV-based Ebola vaccine (rVSV-ZEBOV) expressing the Zaire Ebola virus glycoprotein in a double-blinded study in comparison to a previous study with a high dose (5 × 107 pfu). No serious adverse events occurred and the overall safety was good. The low-dose immunization improved early tolerability, but generated inferior antibody responses and failed to prevent vaccine-induced arthritis, dermatitis or vasculitis. Furthermore, VSV particles expressing the HIV-1 gag gene were evaluated in a clinical trial on safety and immunogenicity. In the randomized double-blinded placebo-controlled dose-escalation study, healthy HIV-negative volunteers received 4.6 × 103 to 3.4 × 107 pfu of rVSV HIV-1 gag vaccine intramuscularly at months 0 and 2. All vaccinated individuals showed antibody responses against VSV, and gag-specific T-cell responses were detected in 63%. Overall, the safety profile was good.
Alphaviruses have been subjected to some gene therapy and vaccine studies. In one approach, replication-deficient SFV particles were encapsulated in liposomes to promote passive targeting of tumors. Initially, intraperitoneal administration of encapsulated SFV-LacZ particles showed enhanced accumulation of β-galactosidase in SCID mice implanted with LNCaP prostate tumors. Liposome-encapsulated SFV particles expressing the p40 and p35 subunits of IL-12 generated active secreted IL-12 in BHK-21 cells. Next, encapsulated SFV-IL-12 particles were administered intravenously in terminally ill melanoma and kidney carcinoma patients in a phase I clinical trial. The patients showed a five to ten-fold increase in IL-12 plasma levels. The maximum tolerated dose was determined to 3 × 109 infectious particles and the safety profile was good. A phase I dose-escalation trial was conducted in prostate cancer patients with VEE particles expressing PSMA. Patients with castration-resistant metastatic prostate cancer (CRPC) received up to five doses of either 0.9 × 107 IU or 0.36 × 108 IU of VEE-PSMA particles at weeks 1, 4, 7, 10 and 18. The study showed no toxicity and good toleration of the vaccination. However, only weak PSMA-specific immune responses were detected and no clinical benefits obtained. In another clinical trial, VEE particles expressing the CEA tumor antigen were demonstrated to efficiently infect DCs. The VEE particles could be repeatedly administered and overcame high titers of neutralizing antibodies and elevated regulatory T cells (Tregs), which allowed induction of clinically relevant CEA-specific T cell and antibody responses. In another approach, VEE particles expressing the cytomegalovirus (CMV) gB and pp65/IE1 fusion protein were evaluated in a phase I randomized, double-blinded clinical trial. Intramuscular or subcutaneous immunization at weeks 0, 8 and 24 of CMV seronegative adult volunteers showed good tolerance with only mild to moderate local reactions and no clinically important changes. Neutralizing and multifunctional T-cell responses against CMV antigens were detected in all vaccinated individuals.
Alphaviruses have found frequent applications in the area of tumor vaccine development (Table 3). In this context, naked RNA, replication-deficient particles and DNA layered vectors have been employed as delivery vehicles. For instance, mice have been subjected to immunization with naked SFV RNA replicons carrying the LacZ gene. Interestingly, a single injection of only 1 μg of SFV‑LacZ RNA presented complete tumor protection. Furthermore, when tumors were administered two days prior to the immunization, the survival was extended by 10–20 days. Among DNA-based tumor vaccine approaches, SIN vectors expressing mouse and human tyrosine-related protein-1 (TRP-1) were evaluated in a B16 mouse melanoma model. Intramuscular injection was capable of breaking immune tolerance and provided protection against melanoma when mice were vaccinated five days prior to cancer challenge. In another study, alphavirus replicon-based expression of melanoma differentiation antigen (MDA) tyrosine demonstrated the inhibition of the growth of B16 transplantable melanoma. In this context, the vaccine encoding tyrosine related protein 2 (TRP-2) relied on a novel immune mechanism, which required the activation of both IgG and CD8+ cell effector responses.
Furthermore, vaccination with recombinant particles expressing the P1A gene and the human papilloma virus (HPV) E7 gene from SFV and VEE vectors, respectively, provided protection against further tumor development in mice. Attempts have also been made to improve the efficacy of SFV-based HPV vaccines by supplying SFV-based IL-12 expression in mice. At low doses, IL-12 stimulated antigen-specific CTL responses and enhanced anti-tumor responses after SFV-based HPV16-E6E7 immunization. Subsequent increases in dosage, however, neither improved the immune responses, nor tumor regression. SIN DNA vectors have been employed for the expression of the murine melanoma cell adhesion molecule (MCAM/MUC18) as a vaccine against murine melanoma, which resulted in the induction of humoral and CD8+ T-cell immune responses against melanoma.
In the context of breast cancer, a DNA-based SIN vector expressing the neu gene was applied for intramuscular vaccination of mice 14 days prior to the injection of cancer cells overexpressing neu. The immunization provided strong protection against tumor development. The incidence of lung metastasis from mammary fat pad tumors was reduced. Moreover, the number of lung metastases from intravenous injection of neu overexpressing cells decreased. Additionally, intradermal vaccination provided tumor protection applying 80% less plasmid than required for conventional DNA vectors. Further confirmation of successful cancer vaccination was obtained from the administration of SIN vectors expressing neu (pSINCP/neu) in a murine breast tumor model. However, in this case, the prophylactic vaccine only showed efficacy when administered prior to the tumor challenge. Another approach was comprised of combining alphavirus-based delivery with the chemical anticancer agent, doxorubicin. When pSINCP/neu DNA and VEE/neu particles were administered after injection of 5 mg/kg of doxorubicin, the tumor progression was significantly delayed. This phenomenon did not occur for doxorubicin alone. Similarly, a combination therapy with paclitaxel (25 mg/kg) and pSINCP/neu was ineffective. Moreover, VEE-neu particles were subcutaneously administered in a rat mammary tumor model, which resulted in the elimination of 36% of pre-existing aggressive mammary tumors. The combination of dendritic cell (DC)-based cancer immunotherapy with VEE-neu particle administration induced both cellular and humoral immunity against neu in transgenic human breast tumor-bearing mice. Moreover, this treatment resulted in the significant inhibition of tumor growth. Similarly, both tumor growth and pulmonary metastatic spread were significantly inhibited when mice with pre‑existing tumors were subjected to five immunizations with SFV10-E VLP expressing the vascular endothelial growth factor receptor 2 (VEGFR-2). Furthermore, co-immunization with SFV particles encoding VEGFR-2 and IL-4 generated significant tumor regression in mice.
Lung cancer has also been targeted by combined therapy with SFV-IL-12 particles and anti-CD137 monoclonal antibodies. Syngeneic TC-1 lung carcinoma was inhibited after intratumoral SFV-IL-12 administration and co-stimulation with anti-CD137 mAbs. In the context of colon cancer vaccines, a SIN-based DNA vector carrying the LacZ gene was compared to conventional plasmid DNA vectors in mice with CT26.CL25 tumors. Intramuscular immunization elicited immune responses at doses 100- to 1,000-fold lower for the SIN DNA replicon vector compared to the conventional CMV‑promoter-based DNA-LacZ vector. Similarly, SFV particles providing VEGFR-2 expressing in vaccinated mice inhibited CT26 colon carcinoma growth. Closer analysis of microvessel density demonstrated that a significant inhibition of tumor angiogenesis occurred. Additionally, when mice were co-immunized with SFV-VEGFR-2 and SFV-IL-4 particles, their survival rate was significantly enhanced. Furthermore, oncolytic SFV vectors have been used for immune stimulation in a CT26 colon tumor model. Intratumoral injections led to an immediate and intense inflammatory reaction and a significant improvement in survival rates. SFV particles expressing HPV16 E6 and E7 have been subjected to prophylactic vaccine development in a murine TC-1 model for cervical cancer. Pre‑immunization with a low dose (104 particles) resulted in an HPV-specific CTL response in 50% of mice, whereas a higher dose (106 particles) elicited CTL responses in all animals. Furthermore, at a dose of 5 × 106 particles, 40% of mice were protected from tumor challenges. In another study, the SFV-enhE6,7 particle vaccine showed its potential after intravenous and intramuscular delivery, where exponentially growing tumors completely resolved. Similarly, SIN virus RNA replicons expressing HPV E7 were evaluated in a TC-1 mouse model. The humoral and cellular immune responses were poor, and no tumor protection was obtained; but, when the HPV E7 gene was fused to the secretory Sig protein and lysosome-associated membrane protein 1 (LAMP-1), enhanced E7‑specific CD4+ helper T-cell and CD8+ cytotoxic T-cell activity was observed. Moreover, strong in vivo anti-tumor activity was induced. In another study, SIN virus particles expressing both HPV E7 and calreticulin (CRT), an endoplasmic reticulum Ca2+ binding transporter, were tested as prophylactic vaccines. Vaccinations generated antigen-specific immune responses, an anti‑angiogenic effect and a strong anti-tumor activity. Furthermore, intramuscular immunization one week prior to challenge with TC-1 carcinoma cells provided protection to all treated mice. Also VEE particles expressing HPV E7 were subcutaneously injected in mice two weeks prior to cancer cell inoculation, which prevented tumor formation. Furthermore, vaccination induced long-term memory, as protection was observed for challenges three months after the immunization. The therapeutic efficacy was only 67% of treated tumor-bearing mice. However, co-expression of HPV E6 and E7 from the same vector significantly enhanced the therapeutic effect.
In a prostate tumor model, VEE particles expressing human prostate-specific membrane antigen (PSMA) showed strong cellular and humoral immunity after subcutaneous administration. Furthermore, VEE particles have been employed for the expression of the predominantly prostate tissue-specific six transmembrane epithelial antigen of the prostate (STEAP). Pre‑immunization with VEE-STEAP particles induced a specific immune response and significantly prolonged the overall survival of mice bearing TRAMPC-2 tumors. When TRAMP mice were prophylactically immunized with a prostate stem cell antigen (PSCA) DNA plasmid followed by VEE‑PSCA particle administration, a specific immune response and anti-tumor protection were observed in 90% of vaccinated animals. Several vaccine studies have targeted brain tumors. For instance, SFV particles expressing endostatin showed a significant reduction of intratumoral vascularization after intratumoral delivery. In another approach, bone-marrow isolated dendritic cells (DCs) were transduced with SFV vectors carrying cytokine genes of specific cDNAs from melanoma and glioma cells. Pre-vaccination with DCs transduced with SFV-based B16 and 203 glioma cDNAs, respectively, resulted in tumor challenge protection and the prolonged survival of tumor-bearing mice. The combination of DCs transduced with SFV-IL-12 particles and systemically administered IL-18 also provided increased survival rates. Moreover, the expression of human melanoma-associated antigen gp100 and IL-18 from a SIN virus DNA vector induced specific anti-tumor CTL responses and provided anti-tumor protection. Vaccination prevented B16-hgp100 tumor formation and demonstrated significant prolongation of survival in mice with established B16-hgp100 tumors.
In summary, numerous preclinical and clinical studies have confirmed the feasibility of the approach of applying self-replicating RNA viruses for both preventive and therapeutic use for various diseases (Table 1, Table 2 and Table 3). In this context, immunization with self-replicating RNA viruses has generated strong immune responses and in many cases provided protection against challenges with lethal doses of infectious agents. Moreover, administration of self-replicating RNA viral vectors expressing anticancer, toxic and/or immunostimulatory genes have demonstrated tumor growth inhibition, regression and even complete tumor eradication, which has supported significant prolongation of survival profiles. Immunization has also provided prophylactic protection against challenges with tumor cells in animal models. One interesting aspect of applying self-replicating RNA viruses comprises of the possibility of using RNA replicons, replication-deficient and -competent particles, and layered DNA/RNA vectors. It provides certain flexibility in choosing the means of delivery vehicle for specific applications. Moreover, several attempts have been made to engineer vectors specifically targeting, replicating and killing tumor cells. One approach has been to apply oncolytic viral vectors, which has provided specific killing of tumor cells without affecting normal cells. In another approach, recombinant SFV particles were encapsulated in liposomes, which provided passive targeting of tumors and protection against recognition by the host immune system. Moreover, the limitation of vector use due to host immune responses has been addressed by engineering a polymer-coated MV-NPL vector based on the MV Edmonston strain with the N, P, and L genes of the wild-type MV strain. The polymer-coated MV-NPL showed superior oncolytic activity in vitro compared to naked MV-NPL. Moreover, polymer-coated MV-NPL provides higher complement-dependent cytotoxicity and antitumor activities than naked virus in mice. In the context of optimization of immune responses, specific targeting of DCs has proven a useful approach demonstrating enhanced immune responses from VEE vectors transducing DCs. Recently, the delivery to DCs and translation of replicon RNA from classical swine fever virus (CSFV) encoding influenza virus NP, belonging to flaviviruses, was improved by lipid formulations, which was demonstrated both in vitro and in vivo by induced immune responses against influenza NP. Moreover, potential enhanced therapeutic efficacy has been addressed by various applications of combination therapy. For instance, a triple treatment combination of sunitinib, low-dose irradiation, and SFV-HPV E6,7 particles rendered mice tumor-free. Similarly, combining VSV immunization with ruxolitinib administration enhanced responses in both subcutaneous and orthotropic xenograft models. Furthermore, ruxolitinib and Polybrene or DEAE-dextran rendered VSV-resistant cells susceptible, which should aid VSV-based therapy.
Although a relatively small number of clinical trials have been conducted with self-replicating RNA viruses, there has been some promising results. Especially, several phase III trials on MV-based vaccines against EBV have provided good safety profiles and protection. Alphavirus vectors have been subjected to clinical trials on infectious diseases. So far, elicited immune responses have been relatively modest, which at least to some extent has been related to lack of dose optimization. In the context of using self-replicating RNA viruses for cancer therapy, less progress has been seen compared to infectious diseases. However, VEE-CEA particles showed prolonged survival in a phase I trial in pancreatic cancer. Moreover, promising results were obtained for liposome encapsulated SFV particles (LipoVIL12) in terminally ill melanoma and kidney carcinoma patients. Also, MV vectors have shown regression of lymphoma lesions, stable disease in treatment of ovarian cancer, and complete response in one myeloma patient.
One important issue related to the utilization of any delivery system is safety. In the first phase, it is essential to provide high safety standards during laboratory research and large-scale production to ensure the protection of personnel. Related to self-replicating RNA viruses, special attention has been paid to the engineering of helper virus vectors used for virus preparation both at laboratory and large-scale. Initially, introduction of point mutations in the p62 precursor of the SFV E2 and E3 envelope genes rendered generated recombinant particles conditionally infectious requiring an additional activation step with α-chymotrypsin. This second generation pSFV-Helper2 vector reduced the generation of replication-competent SFV particles to undetectable levels. Introduction of split helper systems for both SFV and SIN in which the capsid and envelope proteins are placed on separate plasmids generating high-titer particles eliminated production of recombinant-proficient alphavirus particles. Moreover, self-replicating RNA viruses—including alphaviruses, flaviviruses, MVs, and rhabdoviruses—have been classified at laboratory biosafety level 2 although the gene of interest expressed from the vector might impact the level. Related to toxicity and adverse events observed in patients subjected to viral injections, VEE particles were well-tolerated, showed only local reactogenicity, and no clinically important changes. However, although five serious adverse events were recorded in a phase I study in healthy HIV-uninfected individuals none were considered related to the vaccine. The only adverse events related to immunization of Ebola patients with VSV vectors comprised of pain at the injection site, fatigue, myalgia, and headache. Similarly, immunization of healthy volunteers with the VSVΔG-ZEBOV-GP vaccine showed only mild to moderate self-limited adverse events and injection-site pain and headache during a 14-day follow-up period. Related to toxicity issues, a phase I study in CRPC patients demonstrated that VEE-PSMA administration was well-tolerated and no toxicity was observed. Likewise, was well-tolerated in ovarian cancer patients showing no dose-limiting toxicity, MV-CEA. Interestingly, a phase I trial in pancreatic cancer patients showed the feasibility of repeated injections. Moreover, liposome-encapsulated SFV-IL12 particles could be repeatedly administered to kidney carcinoma and melanoma patients without demonstrating any toxicity, or virus- or liposome-related immune responses.
Looking into the future, continuous vector development aiming at delivery and safety improvements will certainly support the progress in therapeutic applications of self-replicating RNA viral vectors. Moreover, dose optimization studies, especially at the clinical level, needs to be conducted. As vaccine development and gene therapy approaches have taken giants leaps recently with classical approaches and more pioneering efforts using viral vectors and nucleic acids. The attractive features of self-replicating RNA viruses relate to the easy of virus production, broad host range, high safety levels due to no risk of chromosomal integration, targeting of DCs, but most importantly the extreme RNA replication in the cytoplasm, which supports high level transgene expression as the basis for generating strong immune responses. Today, RNA-based delivery provides an attractive approach, especially combined with either polymer- or liposome-based encapsulation strategies.
CHIKF outbreak control is hampered by the lack of licensed vaccines that can be used in preventive immunization programs and for emergency response. Although the technical challenges of developing a CHIKF vaccine are not as great as those for some other viral diseases such as dengue, in which multiple serotypes must be targeted and partial immunity can lead to disease enhancement, there are several critical obstacles and financial constraints that need to be overcome in order to make available an affordable and effective vaccine.
First of all, barriers to the acquisition of human efficacy data for vaccine candidates due to the unpredictable nature of CHIKF epidemics may delay the development of a vaccine, even though promising candidates are available. For example, randomized controlled trials, which are considered the gold-standard for evaluating vaccine efficacy, may not be feasible during interepidemics periods because of the low expected number of cases; for this reason, epidemic events with a large number of cases may represent unique opportunities to ensure study power for testing vaccine candidates in efficacy trials. Approaches to overcoming these barriers include the development of platform technologies in which the critical antigens of a newly emerging viral strain can be rapidly incorporated into DNA or RNA platforms with proven safety records. However, based on initial results with DNA Zika vaccines that were generated within a few months of the recognition of congenital Zika Syndrome, these vaccines require multiple doses and immunity is not long lived; they are therefore far from ideal for intervening during an explosive epidemic or for long-term protection in endemic locations. Viral-vectored platforms such as vesicular stomatitis or measles can also be rapidly adapted for new viral targets and may offer more rapid and durable immunity.
Another approach to overcoming challenges of unpredictably emerging viral diseases is acceleration of clinical testing of new vaccine candidates and providing a robust rationale for particular trial designs and regulatory pathways. Therefore, vaccine trials should be designed very carefully to implement quickly and maximize their results during outbreaks. Potential opportunities for CHIKF vaccine testing may include cities with histories of recurrent dengue outbreaks (dengue and CHIKV share the same human-amplified transmission cycles, so regions susceptible to one should eventually have outbreaks of the other); for example, Sao Paulo State, Brazil, and Iquitos, Peru, have still not experienced major CHIKF outbreaks. However, ethical concerns with placebo-immunizing at-risk populations during an epidemic may need to be overcome with nontraditional designs, such as that used during the Ebola vaccine trials in West Africa.
There may also be opportunities to perform efficacy trials in regions endemic for CHIKF, but the typical misdiagnosis of CHIKF as dengue fever will need to be overcome with improved surveillance and diagnostics to identify such opportunities. Finally, CHIKF will continue to occur mainly in poor-resource countries located in tropical areas, where the presence of trained and well-equipped clinical sites, which are essential for the implementation of clinical trials, can be challenging. However, there may be opportunities to capitalize on sites already developed for dengue vaccine trials, which are generally in locations endemic for both viruses.
In case these challenges to clinical efficacy trials cannot be overcome, alternative strategies should be considered. For example, it will be important to obtain reliable information on correlates of immune protection, which are essential in order to apply the so-called “animal rule.” This entails the use of surrogate end-points derived from animal data instead of the results of human trials. This approach could be considered as an alternative option when large efficacy studies on humans, which are usually requested for traditional regulatory approval, are virtually impossible to realize. For example, if human antibodies against CHIKV developed from individuals vaccinated in phase 1 and 2 trials are transferred to NHPs and are demonstrated to confer protection, this could provide surrogate evidence of vaccine efficacy, leading to a provisional license. In this regard, the level of neutralizing antibodies has been already proposed for use as a surrogate marker of vaccine-induced protection [10, 36, 49, 58]. Although there is strong evidence that neutralizing antibodies against CHIKV protect against infection and disease, the lack of compete understanding of chronic arthritis, and its determinants, could limit the ability to relate animal efficacy to human protection. Further work to model arthralgia and arthritis in NHPs could greatly enhance the value of preclinical CHIKF vaccine testing. Other regulatory considerations, including “traditional approval,” “accelerated approval,” and the “animal rule,” have been reviewed extensively in another article focused on CHIKF vaccines.
Obstacles to the provision of scientific evidence are not only represented by the barriers described above to vaccine development. In fact, most research and development (R&D) projects do not deliver a licensed vaccine for routine or targeted immunization—not because of methodological problems, but due to political and economic obstacles. In fact, neglected diseases disproportionally affect poor and marginalized populations, and vaccines may have low returns on investment, so commercial firms may be reluctant to commit themselves to the expensive development and licensure of vaccine candidates, which typically totals hundreds-of-millions of US dollars. To overcome this problem, several strategies may be implemented, including the creation of public and/or private partnerships, the identification of target population groups for vaccination to ensure a potential market, such as the military market, travelers and tourists, and the commitment of donor agencies and affected and/or donor countries. Combination private and public consortia should address those vaccine development projects that are not considered highly profitable by industry in the absence of support from the governments of industrial countries. A recent example is the Coalition for Epidemic Preparedness Innovations (CEPI), funded by both government entities and private foundations, and include partners from the pharmaceutical industry, which is funding late preclinical and clinical development of vaccines for infections by Lassa, Nipah, and MERS coronavirus.
Encephalomyelitis resulting from virus infection of neurons is a disease that can be fatal or result in permanent disability due to irreversible damage of infected neurons. The immune response to infection can enhance neuronal damage or can control virus replication by noncytolytic mechanisms and thus determine outcome. However, noncytolytic virus clearance results in persistence of viral nucleic acid in the CNS and thus establishes a need for long-term local immune responses to prevent reactivation of infection and progressive disease. Understanding these mechanisms is necessary for development of strategies for treating and preventing neurologic disease due to viral encephalomyelitis.
Very little has been done in Indonesia to systematically survey patients admitted to hospitals with CNS infections. Many challenges exist in determining the etiology of viral CNS infections including the timing of specimen collection, optimal storage, locating the laboratory network, and cost of testing. Determining the causes of CNS infections is becoming increasingly important with the emergence of new viruses. With the challenge of taking multiple samples in general hospitals, CSF should be emphasized as the recommended specimen on admission for patients presenting with suspected CNS infection with a follow-up serum specimen at discharge (or death) for serologic confirmation. The results of this study highlight the importance of using a wide range of molecular panels and detection methods for CNS viruses as to optimize treatment strategies, guide public health measures or develop targeted vaccination recommendations. However, in resource-limited settings, it is highly recommended that initial viral screening should include HSV and EV as well as flavivirus serological assays.
In summary, the burden of disease caused by CHIKV is very high, due to the expanding geographic range of virus activity, increasing numbers of cases worldwide, and to the severe and long-lasting arthralgic sequelae of the disease. Developing an effective vaccine is crucial to contain outbreaks and to reduce the clinical and financial impact of CHIKF at the global level. However, as for other neglected and sporadically emerging diseases, barriers to traditional vaccine development and licensure need to be overcome by investing appropriate resources, which may require novel strategies to bring together diverse stakeholders.
Historically, the early version of human vaccines and technologies were developed or discovered based on animals, for example, the smallpox and fowl cholera vaccines. Recent animal vaccine markets can be a good test market to evaluate the new technologies before adopting those new technologies directly to humans. In actual cases, many new vaccine technologies were used for animal vaccines. The most dramatic changes in recent animal vaccines were the usage of VLP-form vaccines for PCV2 and RNA particle vaccines that are produced from gene combinations of vectors and pathogens. As these vaccines are made from the baculovirus in insect cells and the VEE virus, respectively, they can reduce the time for development and make it possible to produce the highly pathogenic vaccine antigens, like the FMD virus, without high-level barrier facilities.
The most important advantage of these vaccine technologies allow us to confront viral diseases, like the influenza virus that is hyper-variable, in a relatively short time. Based on these experiences and technologies for animals, effective preventive tools that are beneficial for human disease can be developed.
Self-replicating RNA viruses have been subjected to a limited number of clinical trials (Table 3). In this context, alphaviruses have been subjected to few clinical trials, so far. For instance, VEE particles expressing CMV gB or a PP65/IE1 fusion protein were applied for a randomized, double-blind phase I clinical trial in CMV seronegative individuals. Intramuscular or subcutaneous administration was well-tolerated with no clinically important changes and direct IFN-γ ELISPOT responses to CMV antigens were detected in all 40 vaccinated subjects. Moreover, immunization elicited neutralizing antibody and multifunctional T cell responses against all three CMV antigens. In another study, healthy HIV-negative volunteers were subjected to double-blind, randomized, placebo-controlled phase I trials in the United States and South Africa applying VEE expressing a nonmyristoylated form of Gag. Subcutaneous administration of VEE-Gag was well-tolerated, but exhibited only modest local immune responses with low levels of binding antibodies and T cell responses. Although five serious adverse events were reported none were considered to be related to the administered vaccine. VEE particles capable of efficiently infecting DCs were employed for the expression of CEA in a clinical trial in patients with advanced cancer. Intramuscular doses of 4 × 107 IU to 4 × 108 IU of VEE-CEA particles were given every three weeks for four immunizations. Repeated immunization induced clinically relevant CEA-specific T cell and antibody responses. The antibody-dependent cellular toxicity against tumor cells from human colorectal cancer metastases were mediated by CEA-specific antibodies. Moreover, longer overall survival was observed in patients with CEA-specific T cell responses. Propagation-defective VEE particles expressing the PSMA have also been subjected to a phase I clinical trial in patients with castration resistant metastatic prostate cancer (CRPC). Five doses of either 0.9 × 107 IU or 3.6 × 107 IU of VEE-PSMA were administered to patients with CRPC metastatic to the bone. Vaccinations were well-tolerated at both doses although only weak PSMA-specific signals were detected. Although neither clinical benefit nor robust immune responses were achieved, the elicited neutralizing antibodies suggest that dosing was suboptimal. In another approach, SFV particles expressing IL-12 (LipoVIL12) were encapsulated in liposomes, which provided passive tumor targeting and protection against host immune recognition. LipoVIL12 was intravenously administered to melanoma and kidney carcinoma patients in a phase I clinical trial. Patients receiving LipoVIL12 showed transient (five days) up to 10-fold increased IL-12 plasma levels. The encapsulation enhanced tumor targeting and prevented host immune recognition after repeated injections. Furthermore, no toxicity was related to the treatment and the maximum tolerated dose (MTD) was determined as 3 × 109 particles per m2.
Several VSV-based clinical trials have been conducted. Two placebo-controlled, double-blind, dose-escalation phase I trials have been performed with recombinant VSV particles expressing the glycoprotein of a Zaire strain of Ebola virus. A total of 78 volunteers received one of three doses (3 × 106, 2 × 107 or 1 × 108 pfu) of VSV-ZEBOV to assess safety and immunogenicity of the vaccination. Some adverse event such as injection-site pain, fatigue, myalgia, and headache occurred. Lower titers were observed at day 28 for the dose of 3 × 106 pfu in comparison to the other two doses.
Furthermore, a second dose at day 28 significantly increased the antibody titers at day 56, but the effect disappeared after 6 months. In another randomized, dose-ranging, observer-blind, placebo-controlled phase I trial, 40 participants received the attenuated VSVΔG-ZEBOV-GP vaccine. No serious adverse events were encountered. All vaccinees developed immune responses comparable across all doses applied. Sustainable IgG titers were detectable throughout the whole study (180 days). Furthermore, another phase I study with VSV-ZEBOV showed good tolerance, no vaccine-related adverse events, and superior cellular immune responses and stronger interlocked cytokine networks for immunization with the highest dose of 2 × 107 pfu. In the Geneva phase I/II, dose-finding, placebo-controlled, double-blind study the VSV-ZEBOV dose was reduced to 3 × 105 pfu compared to previous doses of 1–5 × 107 pfu. The lower dose improved tolerability, but decreased antibody responses. Moreover, it did not prevent vaccine-related arthritis, dermatitis, or vasculitis.
A randomized, placebo-controlled phase III trial was conducted in 1500 adults with the chimpanzee Ad3 (ChAd3-EBO-Z) and the recombinant VSV (rVSV∆G-ZEBOV-GP) vaccines in Liberia. Adverse events including injection-site reactions, headache, fever, and fatigue occurred significantly more frequently in individuals receiving the active vaccine compared to placebo. Antibody responses were detected in 70.8% and 83.7% of subjects in the ChAd3-EBO-Z and the rVSV∆G-ZEBOV-GP groups, respectively, compared to 2.8% in the placebo group one month after vaccination. At 12 months the percentage for the ChAd3-EBO-Z and the rVSV∆G-ZEBOV-GP groups was 63.5% and 79.5%, respectively, with 6.8% in the placebo group. Another phase III trial was conducted in Guinea as an open-label, cluster-randomized ring vaccination study in suspected cases of Ebola virus disease (EBV). A total number of 7651 individuals were included, of which 4123 persons were assigned for immediate vaccination with rVSV-ZEBOV and 3528 persons assigned for delayed vaccination. No cases of EBV were detected in the immediate vaccination group after 10 days, whereas 16 cases of EBV were registered in the delayed vaccination group. No new cases of EBV were diagnosed in either group. Overall, the rVSV-ZEBOV vaccine was confirmed to be safe and showed promise as highly efficient in preventing EBV. Another phase III study was conducted in Guinea and Sierra Leone applying a single intramuscular vaccination with 2 × 107 pfu of rVSV-ZEBOV. In the randomized trial, 2119 individuals were immediately vaccinated and 2041 persons were vaccinated after a delay of 21 days after randomization. Vaccinated individuals were followed up for 84 days offering substantial protection against EBV with no cases of EBV discovered from day 10 after vaccination. Moreover, an individually-controlled phase II/III trial was conducted on health care and frontline workers in the five most EBV affected districts in Sierra Leone. A single intramuscular dose was administered at enrollment or 18–24 weeks after enrollment. The outcome indicated that no EBV cases and no vaccine-related serious adverse events were reported. Finally, a randomized, double-blind, multicenter phase III trial was conducted in the United States, Spain, and Canada.. Vaccination was taken place with doses of 2 × 107 pfu and 1 × 108 pfu of rVSV∆G-ZEBOV-GP and placebo for the assessment of safety and immunogenicity. The vaccine was generally well-tolerated. Although systemic adverse events occurred in comparison to placebo, no vaccine-related severe adverse events or deaths were reported. Overall, the results confirmed the safety of vaccination of the EBV risk population with rVSV∆G-ZEBOV-GP.
MV-Edm vaccine strains have been tested in clinical trials against breast, ovarian, head and neck cancer, glioblastoma, and myeloma. In this context, an open-label, nonrandomized dose-escalation phase I trial was conducted with an unmodified vaccine strain MV-Edm Zagreb (MV-EZ) in patients with cutaneous T cell lymphomas. Intratumor injections of MV-EZ on days 4 and 17 were preceded by subcutaneous IFNα injections (72 and 24 h prior to MV-EZ). The maximum tolerated dose was defined as 103 TCID50. Complete regression of CTCL lesions was observed in one patient, while partial regression was observed in the other patients. Related to recombinant MV, a phase I trial was conducted in patients with advanced ovarian cancer by intraperitoneal injection of MV-CEA. Administration of MV-CEA at doses of 103 to 109 TCID50 confirmed no dose-limiting toxicity. The best objective response comprised stable disease in 14 patients with a median duration of 88 days and a range of 55 to 277 days. All individuals vaccinated with higher dose levels (107–109 TCID50) accomplished stable disease, whereas only five out of 12 patients vaccinated with 103–106 TCID50) achieved it. MV-CEA has also been planned for a phase I clinical trial in patients with recurrent glioblastoma multiforme. The study aims at treatment with a starting dose of 1 × 105 TCID50 of MV-CEA escalating to the maximum dose level of 2 × 107 TCID50. One group of patients will receive direct injections into the resection cavity and in the other group MV-CEA will be administered into recurrent tumors. So far, three patients have received 1 × 105 TCID50 and three other patients 1 × 106 TCID50 in the resection cavity showing no dose-limiting toxicity. Oncolytic MV vectors expressing the human sodium iodide symporter (NIS) have been subjected to a phase I trial. Patients with relapsed refractory myeloma received intravenous MV-NIS or cyclophosphamide two days prior to MV-NIS treatment. The initial dose-escalation study (1 × 106–1 × 109 TCID50) revealed that the MTD was not reached. Therefore, doses of 1 × 1010 and 1 × 1011 TCID50 were tested and the latter dose was planned to be used in a phase II trial. A complete response was observed in one patient treated with 1 × 1011 TCID50. The response persisted for 9 months after which an isolated relapse occurred in the skull without recurrent marrow involvement. Irradiation of the lesion resulted in the patient remaining disease-free for an additional 19 months. Another patient had subjective softening and shrinking of her extramedullary plasmacytomas of her back and thighs.
A total of 74 patients, all from North Sulawesi, were enrolled with a clinical suspicion of CNS infection during the 18-month study period (Fig 1). Details of clinical features, laboratory investigations, and clinical outcomes are presented in Table 1. Forty-nine of the patients (66.2%) were male with a median age of 31 years (range 15–72). Median illness day at admission was 7 days (range 1⎼90), while the median duration of hospital stay was 12.5 days (range 2⎼51). Median duration of hospital stay was 11.5 (range 4⎼38) for deceased patients and 12.5 days (range 2⎼51) for those survived. Twenty-three patients (31.1%) were HIV-positive, while 33 (44.6%) had TB from previous medical history. Most patients (73.0%) had a high-grade fever on admission as well as headache (67.6%), altered consciousness (74.3%), or seizures (37.8%). CT scan results were abnormal in 68.9% of patients (42 out of 61) mostly with meningeal enhancement and focal brain lesions in 36.7% and 20.0% of the patients, respectively.
Only 38 patients who gave consent for lumbar puncture had CSF available in sufficient quantities for viral testing, while 36 patients had only serum and/or throat swabs (Fig 1). The CSF and serum specimens were screened with two arbovirus (alphavirus and flavivirus) and six non-arbovirus degenerate primer sets and by two serological assays (anti-DENV IgM and anti-JEV IgM). In addition, throat swab samples were tested for the six non-arbovirus panels. The viral diagnostic assays performed and viruses identified were summarized in Table 2. A confirmed viral etiology was identified in three (HSV-1, CMV, DENV) (4.1%) and a probable/possible association in 11 (HSV-1, JEV, EBV, EV-D68, RV-A) (14.9%) of enrolled patients. Twelve cases were identified by PCR/RT-PCR while two were identified by serology. The most common viral agent HSV-1 was identified in seven patients (9.5%). Other viruses found were EBV (n = 2, 2.7%), CMV (n = 1, 1.4%), EV-D68 (n = 1, 1.4%), RV-A (n = 1, 1.4%), DENV (n = 1, 1.6%), and JEV (n = 1, 1.6%). There were no viruses isolated from any of the CSF samples. Patients with identified viral infection were found to have lower GCS (median 10.5 [3⎼15] vs 13.5 [7⎼15], P < 0.01) and higher fatality (57.1% vs 20.0%, P < 0.05) than those undiagnosed (Table 1).
In our study with a small sample size, no distinguishable clinical or laboratory characteristics were observed between the different viral agents (Table 3). Five of eight deaths were associated with HSV-1 infection while the remaining three were associated with EBV, CMV, and JEV infections. One HSV-1 and two EBV cases were both HIV-positive and TB history-positive, while one HSV-1 and EV-D68 were HIV-positive only.
Thrombocytopenia, the hallmark of dengue fever was not present in the DENV case on admission and throughout the hospitalization based on serial hematological examination as it might not manifest by the time of neurological presentation. The leukocyte count was slightly high in admission but well within the normal range one week after admission, which was not unusual as peripheral leukocytosis was reported in an earlier study. The patient had a history of fever for five days followed by decreased consciousness and general seizure for three days before admission. Other features of dengue fever including retro-orbital pain, rashes, petechiae, and myalgia were not reported. The patient survived with mild cognitive sequelae on discharge. DENV NS1 antigen, an early marker of DENV infection, was negative in the CSF by using SD Bioline Dengue NS1 Ag rapid test kit (Standard Diagnostics, Inc., South Korea). Serum was not tested for DENV NS1 antigen due to insufficient volume. Furthermore, pan-flavivirus RT-PCR was also negative for both CSF and serum samples.
The fatal JEV case identified in this study was negative for DENV IgM and experienced myalgia for 20 days accompanied by high-grade fever, seizure, altered consciousness, and skin rash for two days prior to hospitalization. CSF sample is not available from this JEV patient and an attempt to isolate the virus from the serum was not successful. Furthermore, the two EV cases (EV-D68 and RV-A) identified in this study presented to the hospital without high-grade fever or respiratory symptoms, although the EV-D68 case suffered multiple generalized seizures for 30 days prior to admission. These EV patients were discharged without any cognitive sequelae.
Self-replicating RNA viruses represented by ssRNA viruses of both negative and positive polarity have been subjected to engineering of efficient gene delivery vectors, which can be applied in the form of recombinant particles, RNA replicons and layered DNA plasmid vectors. In this context, measles (MV), rhabdoviruses, flaviviruses and alphaviruses expressing surface antigens from viruses and other infectious agents have been subjected to immunization studies in animal models. Moreover, similar studies have been conducted with tumor antigens. It seems that MV-, rabies virus (RABV)-, vesicular stomatitis virus (VSV)-, Kunjin virus-, Semliki Forest virus (SFV)-, Sindbis virus (SIN)- and Venezuelan equine encephalitis virus (VEE)-based delivery efficiently elicits humoral and cellular immune responses in immunized animals. Furthermore, numerous cases have demonstrated protection against challenges with lethal viruses/infectious agents or with tumor cells. Most of the studies have been conducted with replication-deficient recombinant particles. However, promising results have also been obtained with layered DNA plasmid vectors. A limited number of studies have applied administration of RNA replicons, but the results have been quite encouraging. The obvious advantage to using nucleic acid-based delivery is the elimination of any risk of virus progeny production through recombination events. On the other hand, superior delivery and prolonged duration of expression can be achieved with recombinant viral particles, especially applying replication-proficient oncolytic viruses. For this reason, it is difficult to make any recommendations related to which delivery format to use, and the choice of target will play an important role in decision making.
Similarly, it is practically impossible to favor one viral vector system over another. Reverse genetics systems engineered for MV and rhabdoviruses and packaging cell lines for flaviviruses surely facilitate recombinant particle production and ease of use. Although packaging cell lines have also been generated for alphaviruses, the straightforward in vitro RNA transcription has provided the means for sufficient preparation of replicon RNA and particles for immunization studies. Obviously, plasmid DNA can be directly applied for vaccinations. In comparison to other viral vectors and also non-viral delivery systems, self-replicating RNA viruses can surely be considered competitive (Figure 5 and Table 4). An extensive comparison to other delivery systems is not within the scope of this review, so only a few examples are addressed. Clearly, adenovirus-based vaccine development and gene therapy has a longer history, which has generated a multitude of vector improvements and also resulted a number of clinical trials. Similarly, herpes simplex virus (HSV) vectors have been frequently applied and HSV-GM-CSF have, for instance, been subjected to phase I−III human clinical trials in glioblastoma and melanoma patients. HSV vectors were recently approved by the FDA for use in standard patient care. Related to non-viral vectors, recently dendrimer-RNA nanoparticles have demonstrated protective immunity against lethal challenges with Ebola virus, influenza H1N1 virus and Toxoplasma gondii after a single injection in BALB/c mice.
Overall, self-replicating RNA viral vectors possess several attractive features. The presence of RNA replicons provides the efficient means for rapid generation of a large number of RNA copies for immediate protein translation in the cytoplasm of host cells. Moreover, the strong subgenomic promoter utilized by alphaviruses generates extreme levels of heterologous gene expression. The transient nature of expression is also an advantage for immunization studies. Furthermore, there is no risk of integration of viral genes in the host genome as the viral RNA is degraded within 3–5 days. In the case of immunization with layered alphavirus DNA vectors, approximately 100- to 1000-fold lower doses are required compared to immunizations with conventional plasmid DNA.
Although strong immune responses have been obtained and protection against challenges with lethal pathogens and tumor cells have been achieved and even tumor regression observed in animals with established tumors, some further technology development is necessary. Much development has been invested in vector design including mutant vectors, enhancement signals, targeting DCs and fusion constructs. Furthermore, quite an effort has been paid to the evaluation of different target antigens and immunogens. Several studies, particularly clinical trials, have indicated that although target-specific immune responses have been obtained, further investment is required in finding the right dose for the achievement of optimal response. One area which recently has received much attention is combination therapy. Tumor-associated antigens (TAAs) have been combined with cytokines and antibodies, as well as drugs and radiation co-administered with cytokines. Additionally, optimization of adjuvant composition and stability issues in case of RNA delivery needs to be addressed. Further research in these areas will certainly provide progress and should make immunotherapy an important approach in both prophylactic and therapeutic applications.
In total, the study included 78 patients with a history of sudden onset of fever, headache, fatigue, nausea, vomiting, rash, myalgia and severe and very painful polyarthralgia suggestive of CHIK infection. Fifty-eight plasma samples were provided by the Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand (Ethical approval ID: COA. No. MU-IRB 2010/251.3108). Twenty CHIKV positive sera samples collected from patients suspected to be infected by CHIKV during routine medical examination, were provided by French National Reference Center for Arboviruses, Marseille, France. Patients had given oral consent according to the national ethical regulations. All used samples were handled anonymously.
In May 2013, porcine epidemic diarrhea (PED) case was reported in the United States for the first time. As PED is an acute and very contagious disease, it caused significant economic loss to the swine industry. Pregnant sows infected with the PED virus during the first 30 days of gestation had a 12.6% decrease of farrow rate (from 91.1% to 78.5%). The number of piglets born alive decreased from 10.7 to 8.5 (piglets/litter) in gilts' litters. The clinical signs and histological characteristics of PED are explained in Fig. 4. As PED had not broken out in North America before, unfortunately there were no vaccines available in the US animal medicine market.
One US venture company, Harris Vaccine Inc., employed alphavirus vector technology to produce a PED vaccine. In June 2014, a new PED vaccine became the first PED virus vaccine to receive a conditional US Department of Agriculture license since the first PED outbreak (Fig. 5). Alphavirus vector technology was used for cluster IV H3N2 influenza vaccines without adjuvants.
Alphaviruses belong to the family Togaviridae. The members of Alphavirus possess a single-stranded RNA with the nucleocapsid surrounded by a membrane protein. Among 26 currently recognized members, Venezuelan equine encephalitis (VEE) virus, which causes epidemics in horses and humans, was chosen as the vaccine vector. Attenuated VEE strains were used for high-level heterologous gene expression vectors as a naked RNA vector, replication deficient recombinant particles, and layered DNA vectors. For an emergency PED vaccine, replication deficient recombinant particles were used resembling the VLP form.
Various viral structural proteins including the influenza virus are targeted for vaccine development with alphavirus vectors.
The alphavirus vector vaccine has shown its efficacy in protection against challenges with the H5N1 virus in chickens. The alphavirus vector has shown cellular or humoral responses and protection against lethal dose virus challenges, non-viral pathogens (like bacteria), and even cancers. Additionally characteristics of RNA particle vaccines based on the alphavirus vector were explained in Table 1.
Primarily, the alphavirus vector vaccine can make it possible for vaccinations in emergency situations. From US PED cases, it was possible to produce the PED vaccine in 13 months by substitution of the interest gene into the alphavirus vector. For a highly pathogenic influenza outbreak, to produce an emergency vaccine is indispensable, but, with traditional vaccine manufacturing methods like virus isolation and inactivation, it is too time-consuming. Therefore, recombinant vaccine technology, like the RNA particle vaccine, can be helpful tools for the prevention of various infectious diseases.
Viruses in the genus Alphavirus belong to the group IV Togaviridae family and include nearly 30 virus species. Alphaviruses are able to infect humans and various vertebrates via arthropods, such as mosquitoes. The 11–12 kb Alphavirus genome is a single-stranded positive sense RNA flanked by a 5’ terminal cap and 3’ poly-A tail, and composed of four non-structural proteins genes (nsP1 to nsP4) and five structural proteins gene (C (nucleocapsid), E3, E2, 6 K, and E1 proteins). Getah virus (GETV) is a mosquito-borne enveloped RNA virus belonging to the Semliki Forest virus (SFV) complex in the genus Alphavirus. To date, 10 strains of GETV have been isolated in China: M1, HB0234, HB0215-3, YN0540, YN0542, SH05-6, SH05-15–17 and GS10-2. GETV has been shown to cause illnesses in humans and livestock animals and antibodies to GETV have been detected in many animal species worldwide.
The identification of novel virus species is important for the identification and characterization of disease. However, present research methods are mostly applicable for known viruses but few methods exist to characterize unknown viruses. Current molecular biological techniques for the identification of new virus species are troublesome since some viruses do not replicate in vitro but some may cause a cytopathic effect. Furthermore, specific techniques that require sequence identification are not applicable. To overcome these limitations, we developed a new method for virus discovery: Virus-Discovery-cDNA RAPD (VIDISCR), based on the cDNA-random amplified polymorphic DNA technique (cDNA-RAPD). VIDISCR includes two key steps. First, the virus genome nucleic acid must be isolated without cellular RNA and DNA contamination. Second the RAPD analysis using the virus genome cDNA or DNA. Using this method, we tested known viruses (SV40 and SV5) and identified a new Getah virus YN08 strain. Virus nsP3, capsid protein genes, and 3’-UTR sequences were cloned, sequenced, and compared. The phylogenetic analysis indicated that the virus YN08 isolate is more closely related to Hebei HB0234 strain than the YN0540 strain, and genetically distant to the MM2021 Malaysia primitive strain.
Human embryonic kidney 293T (HEK293T; ATCC-CLR-N268) and Vero CCL-81 (ATCC #CCL-81) (ATCC, Manassas, VA, USA) cells were cultured in D10 media: Dulbecco Modified Eagle's Medium (Invitrogen Life Science Technologies, San Diego, CA, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS), 3 mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. Mouse splenocytes were cultured in R10 media: (RPMI1640, Invitrogen Life Science Technologies, San Diego, CA, USA) supplemented with 10% heat-inactivated FCS, 3 mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. All cell types were cultured in incubators set to 37°C and 5% CO2.
Disease incidence for any given island was compared to the average incidence for the whole country, using the z-score test for two population proportions available at https://www.socscistatistics.com/tests/ztest/default2.aspx. P values < 0.05 were considered statistically significant.
CHIKV is treated symptomatically by use of painkillers and anti-inflammatory drugs. As there is no effective specific antiviral drug and currently only experimental CHIKV vaccine development. Laboratory diagnosis of CHIKV is very important for effective outbreak management including clinical management and vector control. Therefore, there is a need for a reliable, rapid and portable diagnostic method, which can be deployed in the field during outbreaks.
In this study, we developed a RT-RPA assay for rapid detection of CHIKV in clinical samples, which can be easily deployed to rural health care centers or used in field investigations. We designed a highly sensitive set of RPA primers and exo-probe, which detect CHIKV down to 80 GC/rxn. This detection limit is slightly higher than the 10 GC/rxn detected by reference real-time RT-PCR methods. However, the sensitivity of RT-RPA was sufficient to detect CHIKV infection in clinical samples from Thailand and France with an overall sensitivity and specificity of 100% in comparison to real-time RT-PCR. Previous studies described high titer viraemia exceeding 109 GC/ml in the acute phase of CF. In our study of 20 CF acute samples, viral load ranged from 1.6x106 -1x1010 GC/ml (1.6x104 -1x108 GC/rxn).
The RT-RPA assay detected a panel of 18 different CHIKV strains of all known three genotypes. There was no cross-reactivity of the RT-RPA assay with tested common alpha- and arboviruses except for ONNV detected with the primer combination (RF+RR3), while the combination RF2+RR2 did not detect the ONNV (S2 Fig). Cross reactivity of CHIKV RT-RPA assay to ONNV could be due to 85% similarity of the genomes of ONNV to CHIKV.
RT-RPA primers RF/RR3 harbour four mismatches in RF and seven mismatches in RR3 to the ONNV sequences (S3 Fig). This seems to reflect the fact that RPA assays have been shown to amplify target genes in the presence of up to nine mismatches. Moreover, the position of the mismatch did not influence the amplification step as in the real-time PCR. However, we chose primer pair RF/RR3 due to its faster amplification (Tt: 3.3) and highly analytical sensitivity (23 RNA copies) in comparison to the more specific primer pair RF2/RR2 (Tt: 3.7 and analytical sensitivity: 4310). If necessary, the latter can be used for the differentiation between CHIKV and ONNV e.g. when used in ONNV endemic regions of Africa whereas the former can be used in Asia, Europe and the Americas without any issue.
The RT-RPA assay using RF/RR3 demonstrated a sensitivity of 100% on CHIKV samples of the recent external quality assessment (EQA) study for molecular diagnosis but also detected one sample containing ONNV. Both real-time RT-PCR tests used in this study showed no cross reactivity to ONNV which indicates superior specificity compared to commercial PCR methods tested in the EQA study, in which 46.2% showed cross reactivity to ONNV.
Two loop-mediated isothermal amplification (LAMP) assays for the detection of CHIKV have been published. The LAMP assay require at least four primers and six binding sites and results can be usually observed visually after 30–60 minutes. Recent LAMP assay developments begin to show shorter run times. In contrast, the RPA assay is much faster (3–15 minutes) and utilized two primers and one probe i.e. three binding sites. In addition, RPA reagents are provided in a lyophilized pellet stable at ambient temperature (25–38°C), which allow independence from the cooling chain.
The RPA is a promising technology to perform molecular assay at point of need. Nevertheless, the RPA primer and probe design is still challenging, as dozens of primers combinations must be tested in order to select a functional one. The current RPA assay protocol requires four pipetting steps, which is still less than the real-time PCR method but further development to miniaturize the assay is required. The cost of test per sample is around 5 USD. Lowering the cost to 1 USD will maximum its use in the affected countries.
The CHIKV RPA assay presented here is a promising tool for CHIKV diagnostics at the point of need. Integration into a multimer or multiplex assay for simultaneous and differential detection of CHIKV, Dengue virus and Zika virus as well as an internal positive control would improve outbreak investigations, since the three viruses induce the same clinical picture upon infection and increasingly co-circulate in many parts of the world. Furthermore, combination with a simple extraction method for allowing isolation of virus or virus-infected cells from the whole blood and simple lysis protocol will maximize its employment at low resource settings.
Serum or plasma samples from patient with suspected DENV infection were collected at Port Vila Central Hospital, Efate, Vanuatu between December 2016 to March 2017. 100 μL of material along with 5 μL of EPC was processed using the EasyScreen Sample Processing Kit, SP001 (Genetic Signatures, Sydney, Australia) according to the manufacturer’s instructions. After bisulphite conversion the samples were extracted on the GS-mini automated nucleic extraction platform using the EasyScreen Sample Processing Kit, SP006 (Genetic Signatures, Sydney, Australia) according to the manufacturer’s instructions.
Mayaro virus is an emerging infectious disease agent endemic in tropical regions of South America, but recent evidence suggests that its range may be expanding into Central America and island nations of the Caribbean Sea [2, 11, 35]. The virus causes an acute febrile illness with symptoms including rash, headache, nausea, and diarrhea that can turn into a debilitating, long-term arthralgia in some patients after acute infection has cleared [36, 37]. There are currently no approved vaccines or therapeutics to combat MAYV disease and spread. Here, we report on the generation and immunogenicity of scMAYV-E, a synthetic, enhanced DNA vaccine encoding a novel consensus-designed sequence of the MAYV-envelope glycoproteins. Immunization of mice with scMAYV-E using enhanced EP delivery induced robust, MAYV-specific humoral and cellular responses. Importantly, these responses can neutralize MAYV infection in vitro and can fully protect susceptible mice from morbidity and mortality following MAYV challenge. The results show that scMAYV-E is a highly immunogenic vaccine candidate that warrants further testing in additional systems and animal models for developing countermeasures against MAYV infection and diseases.
The precise correlates of protection for MAYV have not been defined. A recent one-year longitudinal study of confirmed MAYV-infected individuals in Peru found that infection elicited robust anti-viral immune responses including strong neutralizing antibody responses and secretion of pro-inflammatory immune cytokines including IL-13, IL-7, and VEGF. They also report that the strong neutralizing antibody response was not sufficient to prevent long-term negative outcomes of MAYV infection; however, these humoral responses developed post infection. Studies on related alphaviruses, including CHIKV, strongly suggest that a potent, neutralizing antibody response primarily mediates protection from infection, but non-neutralizing antibodies may contribute to protection as well through alternative effector functions. Post infection, there is likely an important role for cellular immunity that may complement the humoral responses. Further studies will be needed to address this important issue.
The scMAYV-E DNA vaccine elicits both humoral and cellular responses against MAYV and consequently be an important tool to provide comprehensive protection from MAYV infection and disease. Antibodies to MAYV are generated after the initial priming immunization with scMAYV-E, and these responses increase after both one and two boosts in terms of binding capacity and affinity to rE1 and rE2. Immune sera from vaccinated mice was able to detect full-length MAYV envelope in scMAYV-E transfected cells as well as MAYV infected cells. scMAYV-E vaccination of mice was able to induce neutralizing antibodies that can block viral entry and inhibit cell death induced by MAYV infection in human MDMs. Passive transfer of immune sera from scMAYV-E vaccinated mice to susceptible naive IFNAR-/- mice prior to MAYV challenge completely protected animals from illness, further confirming the importance of a strong humoral response for conferring protection from alphavirus infection.
Although the anti-MAYV T cell response appears less important for an immediate protection against MAYV infection, it may still be essential for the prevention of chronic disease by eliminating virus-infected cells. The cellular components induced by the scMAYV-E DNA vaccine target multiple epitopes along the full-length MAYV envelope glycoprotein. The strongest cellular responses were directed to epitopes in the E3+E2 domains of the envelope, whereas the responses to epitopes in the E1 glycoprotein were less robust. The epitope mapping studies using ELISpot assays identified two immunodominant epitopes, ‘LAKCPPGEVISVSFV’ in the E3+E2 domain and ‘GRSVIHFSTASAAPS’ within the E1 domain, providing important and useful reagents for studies of the T cell immune response in this haplotype. Interestingly, adaptive transfer of splenocytes from scMAYV-E immunized mice to susceptible naive IFNAR-/- mice prior to MAYV challenge provided partial protection from weight loss and clinical symptoms of MAYV disease, suggesting that MAYV-specific cellular responses do contribute to protection. In this adoptive transfer experiment, MAYV-specific T cells were not purified or enriched from bulk splenocytes prior to transfer, thus it is possible that the partial protection observed here could be enhanced with a larger dose of antigen-specific T cells.
The immunogenicity of the scMAYV-E DNA vaccine mirrors what we observed in a previous DNA vaccine candidate targeting chikungunya virus (CHIKV-E) which encodes a synthetic consensus sequence of the full-length chikungunya envelope protein. The CHIKV-E vaccine was similarly able to generate humoral and cellular responses directed towards the CHIKV envelope protein, and these responses could protect mice from morbidity and mortality following a CHIKV challenge.
The synthetic DNA vaccines have practical advantages for development including ease of manufacture and stability at warmer temperatures, likely reducing the requirement for a cold chain. They are non-live and non-replicating and do not integrate, thus providing conceptual safety advantages as well. Since DNA vaccine vectors do not induce anti-vector serology, they can be administered multiple times with no loss in potency and without interfering with other vaccine protocols. Such logistical and safety advantages warrant further studies of this vaccine approach, especially pertaining to diseases prevalent in resource-poor tropical settings where MAYV is the most prevalent. To the best of our knowledge, scMAYV-E is the third vaccine candidate for MAYV developed. The first vaccine was an inactivated Mayaro virus, and the second candidate reported was a live-attenuated MAYV vaccine [19, 20]. Both prior vaccines were shown to induce anti-MAYV humoral responses that could protect mice from MAYV challenge, but neither study reported on the induction of cellular responses to MAYV. The synthetic scMAYV-E DNA vaccine described here generates MAYV-specific humoral and cellular responses without viral replication, which is likely important for immune-challenged, young, pregnant, and elderly populations of potential travelers and residents in endemic areas in need of vaccine-induced immune protection. Additional studies of this vaccine approach using the DNA platform will provide further insight into the relative merits of such methods in the field.
In order to examine the broad immunological applicability of the selected scFvs, we also tested the recombinant antibody fragments for the specific detection of VEEV TC83 in lysates of infected Vero cells. These cell lysates were prepared by disrupting infected cells with 4 M urea while coupled to microwells. Detection was performed with scFv phage followed by an incubation with mAb anti-M13 conjugated to HRP. Lysates of non-infected Vero cells and VEEV antigen incubated with the mAb II-B6 served as negative control.
Specific binding could be confirmed for nearly all selected antibody fragments except for the clones CHN24-2-A1 and MK269-E11. The most stringent binding results were obtained with the scFv clones CHN24-2-A2, CHN24-2-C3, CHN24-2-F11 and MK271-G2 (Fig. 7).
In addition, detection of VEEV-specific antigen by immunohistochemistry in TC83 infected and formaldehyde fixed Vero cells was possible. Similar to the results described above, all scFv clones, except for clone CHN24-2-A1, showed a specific cytoplasmic immunostaining of VEEV infected Vero cells (data not shown).
Sequences of DENV and WNV E with positive Wimley-White interfacial hydrophobicity scale scores were determined using the program Membrane Protein eXplorer. After consideration of the known secondary structures for several subdomains of E, selected peptides were synthesized by solid-phase conventional N-α-9-flurenylmethyloxycarbonyl chemistry (Genemed Synthesis, San Francisco, CA). Peptides were purified by reverse-phase high performance liquid chromatography and confirmed by amino acid analysis and electrospray mass spectrometry. Peptide stock solutions were prepared in 20% (v/v) dimethyl sulfoxide (DMSO): 80% (v/v) H20, and concentrations determined by absorbance of aromatic side chains at 280 nm. Scrambled peptides sequences were obtained by drawing from a hat.
This study was carried out in strict accordance with the recommendations for care and use of animals by the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health. The Institutional Animal Care and Use Committee (IACUC) of The University of North Carolina at Chapel Hill (UNC; permit number A-3410-01) approved the animal study protocol followed in the manuscript. Animals were anesthetized with ketamine and xylazine (per IACUC, UNC, guidelines) for infection and were euthanized if the body weight dropped below 80% of starting weight or clinical symptoms warranted it per IACUC, UNC, guidelines. UNC is registered with the Centers for Disease Control and Prevention to work with select agents, including SARS-CoV.
For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The described antibody selection procedure may also be useful for the in vitro antibody selection of antibody fragments against other viral targets from human naïve antibody gene libraries, in particular when immunized patients are not available or immunisation is not ethically feasible. The broad and sensitive applicability of anti-VEEV scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected recombinant antibody fragments will improve the rapid and specific detection of VEEV infections after human and equine outbreaks of encephalitis, where an early and definite identification is of critical importance.