Etiologies of Acute Undifferentiated Febrile Illnesses in and near Iquitos from 1993 to 1999 in the Amazon River Basin of Peru.

The objective of this study was to determine the etiology of febrile illnesses among patients from October 1, 1993 through September 30, 1999, in the urban community of Iquitos in the Amazon River Basin of Peru. Epidemiological and clinical data as well as blood samples were obtained from consenting patients at hospitals, health clinics and private residences. Samples were tested for arboviruses in cell cultures and for IgM and IgG antibodies by ELISA. Blood smears were examined for malaria, and sera were tested for antibodies to Leptospira spp. by ELISA and microscopic agglutination. Among 6,607 febrile patients studied, dengue viruses caused 14.6% of the cases, and Venezuelan equine encephalitis virus caused 2.5%, Oropouche virus 1.0%, Mayaro virus 0.4%, and other arboviruses caused 0.2% of the cases. Also, 22.9% of 4,844 patients tested were positive for malaria, and of 400 samples tested, 9% had evidence of acute leptospirosis. Although the study was not designed to assess the importance of these pathogens as a cause of human morbidity in the total population, these results indicate that arboviruses, leptospirosis, and malaria were the cause of approximately 50% of the febrile cases. Although the arboviruses that were diagnosed can produce asymptomatic infections, our findings increased the overall understanding of the relative health burden of these infections, as well as baseline knowledge needed for designing and implementing further studies to better assess the health impact and threat of these pathogens in the Amazon Basin of Peru.

Pseudovirus-Based Assays for the Measurement of Antibody-Mediated Neutralization of SARS-CoV-2.

SARS-CoV-2 has emerged as a significant cause of morbidity and mortality worldwide. Virus neutralization assays are critical for the development and evaluation of vaccines and immunotherapeutics, as well as for conducting basic research into the immune response, spread, and pathogenesis of this disease. However, neutralization assays traditionally require the use of infectious virus which must be carefully handled in a BSL-3 setting, thus complicating the assay and restricting its use to labs with access to BSL-3 facilities. Pseudovirus-based assays are an alternative to the use of infectious virus. SARS-CoV-2 pseudovirus contains only the spike structural protein, and infection results in a single round of replication, thus allowing for the assay to be run safely under BSL-2 conditions. In this chapter, we describe protocols and considerations for the production and titration of lentivirus-based SARS-CoV-2 pseudovirus, as well as for running and analysis of FACS-based pseudovirus neutralization assays.

Enhanced Immunogenicity of Inactivated Dengue Vaccines by Novel Polysaccharide-Based Adjuvants in Mice.

Dengue fever, caused by any of four dengue viruses (DENV1-4), is a major global burden. Currently, there is no effective vaccine that prevents infection in dengue naïve populations. We tested the ability of two novel adjuvants (Advax-PEI and Advax-2), using aluminum hydroxide (alum) as control, to enhance the immunogenicity of formalin- or psoralen-inactivated (PIV or PsIV) DENV2 vaccines in mice. Mice were vaccinated on days 0 and 30, and serum samples were collected on days 30, 60, 90, and 101. Neutralizing antibodies were determined by microneutralization (MN) assays, and the geometric mean 50% MN (MN50) titers were calculated. For the PIV groups, after one dose MN50 titers were higher in the novel adjuvant groups compared to the alum control, while MN50 titers were comparable between the adjuvant groups after the second dose. For the PsIV groups, both novel adjuvants induced higher MN50 titers than the alum control after the second dose. Spleen cells were collected on days 45 and 101 for enzyme-linked immunospot (ELISPOT) for IFNγ and IL4. Both PIV and PsIV groups elicited different degrees of IFNγ and IL4 responses. Overall, Advax-2 gave the best responses just ahead of Advax-PEI. Given Advax-2's extensive human experience in other vaccine applications, it will be pursued for further development.

Immunogenicity of Adjuvanted Psoralen-Inactivated SARS-CoV-2 Vaccines and SARS-CoV-2 Spike Protein DNA Vaccines in BALB/c Mice.

The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are over 160 SARS-CoV-2 vaccine candidates at the clinical or pre-clinical stages of development. Of these, there are only three whole-virus vaccine candidates produced using ß-propiolactone or formalin inactivation. Here, we prepared a whole-virus SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) using a novel psoralen inactivation method and evaluated its immunogenicity in mice using two different adjuvants, alum and Advax-2. We compared the immunogenicity of SARS-CoV-2 PsIV against SARS-CoV-2 DNA vaccines expressing either full-length or truncated spike proteins. We also compared the psoralen-inactivated vaccine against a DNA prime, psoralen-inactivated vaccine boost regimen. After two doses, the psoralen-inactivated vaccine, when administered with alum or Advax-2 adjuvants, generated a dose-dependent neutralizing antibody responses in mice. Overall, the pattern of cytokine ELISPOT responses to antigen-stimulation observed in this study indicates that SARS-CoV-2 PsIV with the alum adjuvant promotes a Th2-type response, while SARS-CoV-2 PsIV with the Advax-2 adjuvant promotes a Th1-type response.

Tetravalent dengue DNA vaccine is not immunogenic when delivered by retrograde infusion into salivary glands.

INTRODUCTION AND BACKGROUND: A tetravalent DNA vaccine for Dengue virus is under development but has not yet achieved optimal immunogenicity. Salivary glands vaccination has been reported efficacious in rodents and dogs. We report on a pilot study testing the salivary gland as a platform for a Dengue DNA vaccine in a non-human primate model. MATERIALS AND METHODS: Four cynomolgus macaques were used in this study. Each macaque was pre-medicated with atropine and sedated with ketamine. Stensen's duct papilla was cannulated with a P10 polyethylene tube, linked to a 500ul syringe. On the first two infusions, all macaques were infused with 300ul of TVDV mixed with 2 mg of zinc. For the 3rd infusion, to increase transfection into salivary tissue, two animals received 100uL TVDV mixed with 400uL polyethylenimine 1µg/ml (PEI) and the other two animals received 500uL TVDV with zinc. Antibody titers were assessed 4 weeks following the second and third infusion. RESULTS AND CONCLUSIONS: SGRI through Stensen's duct is a well-tolerated, simple and easy to reproduce procedure. TVDV infused into macaques salivary glands elicited a significantly weaker antibody response than with different delivery methods.

Comparison of purified psoralen-inactivated and formalin-inactivated dengue vaccines in mice and nonhuman primates.

Dengue fever, caused by dengue viruses (DENV 1-4) is a leading cause of illness and death in the tropics and subtropics. Therefore, an effective vaccine is urgently needed. Currently, the only available licensed dengue vaccine is a chimeric live attenuated vaccine that shows varying efficacy depending on serotype, age and baseline DENV serostatus. Accordingly, a dengue vaccine that is effective in seronegative adults, children of all ages and in immunocompromised individuals is still needed. We are currently researching the use of psoralen to develop an inactivated tetravalent dengue vaccine. Unlike traditional formalin inactivation, psoralen inactivates pathogens at the nucleic acid level, potentially preserving envelope protein epitopes important for protective anti-dengue immune responses. We prepared highly purified monovalent vaccine lots of formalin- and psoralen-inactivated DENV 1-4, using Capto DeVirS and Capto Core 700 resin based column chromatography. Tetravalent psoralen-inactivated vaccines (PsIV) and formalin-inactivated vaccines (FIV) were prepared by combining the four monovalent vaccines. Mice were immunized with either a low or high dose of PsIV or FIV to evaluate the immunogenicity of monovalent as well as tetravalent formulations of each inactivation method. In general, the monovalent and tetravalent PsIVs elicited equivalent or higher titers of neutralizing antibodies to DENV than the FIV dengue vaccines and this response was dose dependent. The immunogenicity of tetravalent dengue PsIVs and FIVs were also evaluated in nonhuman primates (NHPs). Consistent with what was observed in mice, significantly higher neutralizing antibody titers for each dengue serotype were observed in the NHPs vaccinated with the tetravalent dengue PsIV compared to those vaccinated with the tetravalent dengue FIV, indicative of the importance of envelope protein epitope preservation during psoralen inactivation of DENV.

Enhanced immunogenicity and protective efficacy of a tetravalent dengue DNA vaccine using electroporation and intradermal delivery.

Phase 1 clinical trials with a DNA vaccine for dengue demonstrated that the vaccine is safe and well tolerated, however it produced less than optimal humoral immune responses. To determine if the immunogenicity of the tetravalent dengue DNA vaccine could be enhanced, we explored alternate, yet to be tested, methods of vaccine administration in non-human primates. Animals were vaccinated on days 0, 28 and 91 with either a low (1 mg) or high (5 mg) dose of vaccine by the intradermal or intramuscular route, using either needle-free injection or electroporation devices. Neutralizing antibody, IFN-γ T cell and memory B cell responses were compared to a high dose group vaccinated with a needle-free intramuscular injection delivery device similar to what had been used in previous preclinical and clinical studies. All previously untested vaccination methodologies elicited improved immune responses compared to the high dose needle-free intramuscular injection delivery group. The highest neutralizing antibody responses were observed in the group that was vaccinated with the high dose formulation via intradermal electroporation. The highest IFN-γ T cell responses were also observed in the high dose intradermal electroporation group and the CD8+ T cells were the dominant contributors for the IFNγ response. Memory B cells were detected for all four serotypes. More than a year after vaccination, groups were challenged with dengue-1 virus. Both the low and high dose intradermal electroporation groups had significantly fewer days of dengue-1 virus RNAemia compared to the control group. The results from this study demonstrate that using either an electroporation device and/or the intradermal route of delivery increases the immune response generated by this vaccine in non-human primates and should be explored in humans.

Safety and Immunogenicity of a Tetravalent Dengue DNA Vaccine Administered with a Cationic Lipid-Based Adjuvant in a Phase 1 Clinical Trial.

We conducted an open label, dose escalation Phase 1 clinical trial of a tetravalent dengue DNA vaccine (TVDV) formulated in Vaxfectin® to assess safety and immunogenicity. A total of 40 dengue- and flavivirus-naive volunteers received either low-dose (1 mg) TVDV alone (N = 10, group 1), low-dose TVDV (1 mg) formulated in Vaxfectin (N = 10, group 2), or high-dose TVDV (2 mg, group 3) formulated in Vaxfectin® (N = 20). Subjects were immunized intramuscularly with three doses on a 0-, 30-, 90-day schedule and monitored. Blood samples were obtained after each immunization and various time points thereafter to assess anti-dengue antibody and interferon gamma (IFNγ) T-cell immune responses. The most common adverse events (AEs) across all groups included mild to moderate pain and tenderness at the injection site, which typically resolved within 7 days. Common solicited signs and symptoms included fatigue (42.5%), headache (45%), and myalgias (47.5%). There were no serious AEs related to the vaccine or study procedures. No anti-dengue antibody responses were detected in group 1 subjects who received all three immunizations. There were minimal enzyme-linked immunosorbent assay and neutralizing antibody responses among groups 2 and 3 subjects who completed the immunization schedule. By contrast, IFNγ T-cell responses, regardless of serotype specificity, occurred in 70%, 50%, and 79% of subjects in groups 1, 2, and 3, respectively. The largest IFNγ T-cell responses were among group 3 subjects. We conclude that TVDV was safe and well-tolerated and elicited predominately anti-dengue T-cell IFNγ responses in a dose-related fashion.

NK cell degranulation as a marker for measuring antibody-dependent cytotoxicity in neutralizing and non-neutralizing human sera from dengue patients.

The study assessed antibody-dependent NK cell degranulation, a biomarker relevant to antibody-dependent cell cytotoxicity (ADCC), to analyze dengue immune sera. We first determined binding intensity of patient sera to the surface of DENV-infected cells and examined the types of antigens expressed on infected cells. Antigens from pre-membrane (PreM) and envelope (E), but not from NS proteins were detected on the surface of infected cells. After adding NK cells to infected target cells previously treated with patient sera, rapid NK cell degranulation was observed. Non-neutralizing patient sera generated comparable NK cell degranulation as that of neutralizing sera, suggesting ADCC may be a protective mechanism apart from Ab neutralization. The level of NK cell degranulation varied dramatically among human individuals and was associated with the level of CD16 expression on NK cells, informing on the complexity of ADCC among human population.

DNA Vaccine Delivery and Improved Immunogenicity.

The promise of DNA vaccines is as compelling today as it was more than a decade ago. Ease of manufacture, stability at ambient temperatures without the need for a cold chain and its ability to mimic natural infections and elicit appropriate immune responses makes this vaccine platform extremely attractive. Although, human clinical trials of DNA vaccines have yielded less than optimal results, the approval and licensing of a few veterinary vaccines is testimony to the proof-of-concept and the hope that licensed DNA vaccines for human use may not be too far away. Delivery and targeting of immunologically relevant cells appears to be the major hurdle in maximizing the immunogenicity of DNA vaccines. Several different approaches that are currently pursued in achieving this objective are discussed.

The Epidemiology, Virology and Clinical Findings of Dengue Virus Infections in a Cohort of Indonesian Adults in Western Java.

BACKGROUND: Dengue has emerged as one of the most important infectious diseases in the last five decades. Evidence indicates the expansion of dengue virus endemic areas and consequently the exponential increase of dengue virus infections across the subtropics. The clinical manifestations of dengue virus infection include sudden fever, rash, headache, myalgia and in more serious cases, spontaneous bleeding. These manifestations occur in children as well as in adults. Defining the epidemiology of dengue in a given area is critical to understanding the disease and devising effective public health strategies. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the results from a prospective cohort study of 4380 adults in West Java, Indonesia, from 2000-2004 and 2006-2009. A total of 2167 febrile episodes were documented and dengue virus infections were confirmed by RT-PCR or serology in 268 cases (12.4%). The proportion ranged from 7.6 to 41.8% each year. The overall incidence rate of symptomatic dengue virus infections was 17.3 cases/1,000 person years and between September 2006 and April 2008 asymptomatic infections were 2.6 times more frequent than symptomatic infections. According to the 1997 WHO classification guidelines, there were 210 dengue fever cases, 53 dengue hemorrhagic fever cases (including one dengue shock syndrome case) and five unclassified cases. Evidence for sequential dengue virus infections was seen in six subjects. All four dengue virus serotypes circulated most years. Inapparent dengue virus infections were predominantly associated with DENV-4 infections. CONCLUSIONS/SIGNIFICANCE: Dengue virus was responsible for a significant percentage of febrile illnesses in an adult population in West Java, Indonesia, and this percentage varied from year to year. The observed incidence rate during the study period was 43 times higher than the reported national or provincial rates during the same time period. A wide range of clinical severity was observed with most infections resulting in asymptomatic disease. The circulation of all four serotypes of dengue virus was observed in most years of the study.

Nucleic acid (DNA) immunization as a platform for dengue vaccine development.

Since the early 1990s, DNA immunization has been used as a platform for developing a tetravalent dengue vaccine in response to the high priority need for protecting military personnel deployed to dengue endemic regions of the world. Several approaches have been explored ranging from naked DNA immunization to the use of live virus vectors to deliver the targeted genes for expression. Pre-clinical animal studies were largely successful in generating anti-dengue cellular and humoral immune responses that were protective either completely or partially against challenge with live dengue virus. However, Phase 1 clinical evaluation of a prototype monovalent dengue 1 DNA vaccine expressing prM and E genes revealed anti-dengue T cell IFNγ responses, but poor neutralizing antibody responses. These less than optimal results are thought to be due to poor uptake and expression of the DNA vaccine plasmids. Because DNA immunization as a vaccine platform has the advantages of ease of manufacture, flexible genetic manipulation and enhanced stability, efforts continue to improve the immunogenicity of these vaccines using a variety of methods.

Tetravalent DNA vaccine product as a vaccine candidate against dengue.

Dengue is the most important arbovirus worldwide and is the virus that causes dengue fever and the more severe dengue hemorrhagic fever. There are four serotypes of dengue with each possessing the ability to cause disease. Developing a preventive vaccine is the most efficient and effective way to prevent these diseases, and because immunity to one serotype does not protect against the other serotypes, a vaccine must provide tetravalent protection. We used DNA immunization as a platform to develop a tetravalent vaccine. In this chapter, we describe the laboratory, regulatory, and clinical methodology for evaluating a candidate tetravalent vaccine in a Phase 1 clinical trial.

Evidence for endemic chikungunya virus infections in Bandung, Indonesia.

Chikungunya virus (CHIKV) is known to cause sporadic or explosive outbreaks. However, little is known about the endemic transmission of CHIKV. To ascertain the endemic occurrence of CHIKV transmission, we tested blood samples from patients with a non-dengue febrile illness who participated in a prospective cohort study of factory workers in Bandung, Indonesia. From August 2000 to June 2004, and September 2006 to April 2008, 1901 febrile episodes occurred and 231 (12.2%) dengue cases were identified. The remaining febrile cases were evaluated for possible CHIKV infection by measuring anti-CHIKV IgM and IgG antibodies in acute and convalescent samples. Acute samples of serologically positive cases were subsequently tested for the presence of CHIKV RNA by RT-PCR and/or virus isolation. A total of 135 (7.1%) CHIKV infections were identified, providing an incidence rate of 10.1/1,000 person years. CHIKV infections were identified all year round and tended to increase during the rainy season (January to March). Severe illness was not found and severe arthralgia was not a prominently reported symptom. Serial post-illness samples from nine cases were tested to obtain a kinetic picture of IgM and IgG anti-CHIKV antibodies. Anti-CHIKV IgM antibodies were persistently detected in high titers for approximately one year. Three patients demonstrated evidence of possible sequential CHIKV infections. The high incidence rate and continuous chikungunya cases in this adult cohort suggests that CHIKV is endemically transmitted in Bandung. Further characterization of the circulating strains and surveillance in larger areas are needed to better understand CHIKV epidemiology in Indonesia.

Immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue DNA vaccine.

A prototype dengue-1 DNA vaccine was shown to be safe and immunogenic in a previous Phase 1 clinical trial. Anti-dengue-1 neutralizing antibody responses were detectable only in the group of volunteers receiving the high dose of nonadjuvanted vaccine and the antibody titers were low. Vaxfectin(®), a lipid-based adjuvant, enhances the immunogenicity of DNA vaccines. We conducted a nonhuman primate study to evaluate the effect of Vaxfectin(®) on the immunogenicity of a tetravalent dengue DNA vaccine. Animals were immunized on days 0, 28 and 84, with each immunization consisting of 3mg of Vaxfectin(®)-adjuvanted tetravalent dengue DNA vaccine. The use of Vaxfectin(®) resulted in a significant increase in anti-dengue neutralizing antibody responses against dengue-1, -3 and -4. There was little to no effect on T cell responses as measured by interferon gamma ELISPOT assay. Animals immunized with the Vaxfectin(®)-formulated tetravalent DNA vaccine showed significant protection against live dengue-2 virus challenge compared to control animals (0.75 mean days of viremia vs 3.3 days). Animals vaccinated with nonadjuvanted DNA had a mean 2.0 days of viremia. These results support further evaluation of the Vaxfectin(®)-adjuvanted tetravalent dengue DNA vaccine in a Phase 1 clinical trial.

Development of dengue DNA vaccines.

Vaccination with plasmid DNA against infectious pathogens including dengue is an active area of investigation. By design, DNA vaccines are able to elicit both antibody responses and cellular immune responses capable of mediating long-term protection. Great technical improvements have been made in dengue DNA vaccine constructs and trials are underway to study these in the clinic. The scope of this review is to highlight the rich history of this vaccine platform and the work in dengue DNA vaccines accomplished by scientists at the Naval Medical Research Center. This work resulted in the only dengue DNA vaccine tested in a clinical trial to date. Additional advancements paving the road ahead in dengue DNA vaccine development are also discussed.

Immunogenicity and protective efficacy of a psoralen-inactivated dengue-1 virus vaccine candidate in Aotus nancymaae monkeys.

Psoralens are photoreactive compounds that cross-link pyrimidines after exposure to UVA radiation. In this experiment, we tested the protective efficacy of a psoralen-inactivated dengue vaccine candidate in non-human primates. Two groups of 7 Aotus nancymaae monkeys received either 10ng per dose of inactivated DENV1 plus alum adjuvant or alum alone (controls). Doses were injected intradermally on days 0, 14, and 28. Monkeys then received a challenge inoculation of 1.1 × 10(4)PFUs of WestPac 74 DENV-1 on day 132. At 62 days, only 1/7 vaccinated monkeys had detectable IgM, but IgG and neutralizing antibody remained detectable in 7/7. No IgM, IgG, or neutralizing antibody was detectable in control monkeys. DENV-1 viremia was detected after challenge in 3/7 vaccinated monkeys and 5/6 control monkeys (with one removed due to pregnancy) (p=0.27), but days of viremia were reduced from 3.67 days/animal among controls to 0.71 days/animal among vaccinated monkeys (p=0.051). Psoralen-inactivated DENV1 is immunogenic in Aotus nancymaae with a trend towards a reduction in days of viremia following experimental challenge.

Evaluation of a prototype dengue-1 DNA vaccine in a Phase 1 clinical trial.

Candidate dengue DNA vaccine constructs for each dengue serotype were developed by incorporating pre-membrane and envelope genes into a plasmid vector. A Phase 1 clinical trial was performed using the dengue virus serotype-1 (DENV-1) vaccine construct (D1ME(100)). The study was an open-label, dose-escalation, safety and immunogenicity trial involving 22 healthy flavivirus-naïve adults assigned to one of two groups. Each group received three intramuscular injections (0, 1, and 5 months) of either a high dose (5.0mg, n=12) or a low dose (1.0mg, n=10) DNA vaccine using the needle-free Biojector(®) 2000. The most commonly reported solicited signs and symptoms were local mild pain or tenderness (10/22, 45%), local mild swelling (6/22, 27%), muscle pain (6/22, 27%) and fatigue (6/22, 27%). Five subjects (41.6%) in the high dose group and none in the low dose group developed detectable anti-dengue neutralizing antibodies. T-cell IFN gamma responses were detected in 50% (4/8) and 83.3% (10/12) of subjects in the low and high dose groups, respectively. The safety profile of the DENV-1 DNA vaccine is acceptable at both doses administered in the study. These results demonstrate a favorable reactogenicity and safety profile of the first in human evaluation of a DENV-1 DNA vaccine.

Immunogenicity of a psoralen-inactivated dengue virus type 1 vaccine candidate in mice.

We evaluated a novel psoralen-inactivated dengue virus type 1 (DENV-1) vaccine candidate in Mus musculus mice. Mice received intradermal alum or 5 to 10 ng of psoralen-inactivated virus. Anti-DENV-1 neutralizing antibody was detectable in 10/11 mice receiving a 10-ng dose at 90 days. Psoralen-inactivated DENV-1 is immunogenic in mice.

Functional characterization of ex vivo blood myeloid and plasmacytoid dendritic cells after infection with dengue virus.

Myeloid and plasmacytoid dendritic cells (mDC and pDC) are naturally distinctive subsets. We exposed both subsets to dengue virus (DV) in vitro and investigated their functional characteristics. High levels of DV replication in mDC were found to correlate with DC-SIGN expression. Production of inflammatory cytokines by mDC increased gradually after DV-infection, which was dependent on DV replication. Co-stimulatory markers were upregulated on mDC upon DV-infection. On the contrary, lower levels of DV-replication were observed in pDC, but the cytokine production in pDC was quicker and stronger. This cytokine response was not dependent on viral replication, but dependent on cell endosomal activity and TLR7, and could be also induced by purified DV genome RNA. These results clearly suggested functional differences between mDC and pDC in response to DV infection. Additionally, the TLR7-mediated recognition of DV RNA may be involved in pDC functional activation.