Safety of Streptococcus pyogenes Vaccines: Anticipating and Overcoming Challenges for Clinical Trials and Post-Marketing Monitoring.

Streptococcus pyogenes (Strep A) infections result in a vastly underestimated burden of acute and chronic disease globally. The Strep A Vaccine Global Consortium's (SAVAC's) mission is to accelerate the development of safe, effective, and affordable S. pyogenes vaccines. The safety of vaccine recipients is of paramount importance. A single S. pyogenes vaccine clinical trial conducted in the 1960s raised important safety concerns. A SAVAC Safety Working Group was established to review the safety assessment methodology and results of more recent early-phase clinical trials and to consider future challenges for vaccine safety assessments across all phases of vaccine development. No clinical or biological safety signals were detected in any of these early-phase trials in the modern era. Improvements in vaccine safety assessments need further consideration, particularly for pediatric clinical trials, large-scale efficacy trials, and preparation for post-marketing pharmacovigilance.

An mRNA Vaccine against SARS-CoV-2 - Preliminary Report.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and spread globally, prompting an international effort to accelerate development of a vaccine. The candidate vaccine mRNA-1273 encodes the stabilized prefusion SARS-CoV-2 spike protein. METHODS: We conducted a phase 1, dose-escalation, open-label trial including 45 healthy adults, 18 to 55 years of age, who received two vaccinations, 28 days apart, with mRNA-1273 in a dose of 25 µg, 100 µg, or 250 µg. There were 15 participants in each dose group. RESULTS: After the first vaccination, antibody responses were higher with higher dose (day 29 enzyme-linked immunosorbent assay anti-S-2P antibody geometric mean titer [GMT], 40,227 in the 25-µg group, 109,209 in the 100-µg group, and 213,526 in the 250-µg group). After the second vaccination, the titers increased (day 57 GMT, 299,751, 782,719, and 1,192,154, respectively). After the second vaccination, serum-neutralizing activity was detected by two methods in all participants evaluated, with values generally similar to those in the upper half of the distribution of a panel of control convalescent serum specimens. Solicited adverse events that occurred in more than half the participants included fatigue, chills, headache, myalgia, and pain at the injection site. Systemic adverse events were more common after the second vaccination, particularly with the highest dose, and three participants (21%) in the 250-µg dose group reported one or more severe adverse events. CONCLUSIONS: The mRNA-1273 vaccine induced anti-SARS-CoV-2 immune responses in all participants, and no trial-limiting safety concerns were identified. These findings support further development of this vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; mRNA-1273 ClinicalTrials.gov number, NCT04283461).

Experimental dengue virus challenge of human subjects previously vaccinated with live attenuated tetravalent dengue vaccines.

BACKGROUND: Protection against dengue requires immunity against all 4 serotypes of dengue virus (DENV). Experimental challenge may be useful in evaluating vaccine-induced immunity. METHODS: Ten subjects previously vaccinated with a live attenuated tetravalent dengue vaccine (TDV) and 4 DENV-naive control subjects were challenged by subcutaneous inoculation of either 10(3) plaque-forming units (PFU) of DENV-1 or 10(5) PFU of DENV-3. Two additional subjects who did not develop DENV-3 neutralizing antibody (NAb) from TDV were revaccinated with 10(4) PFU of live attenuated DENV-3 vaccine to evaluate memory response. RESULTS: All 5 TDV recipients were protected against DENV-1 challenge. Of the 5 TDV recipients challenged with DENV-3, 2 were protected. All DENV-3-challenge subjects who developed viremia also developed elevated liver enzyme levels, and 2 had values that were >10 times greater than normal. Of the 2 subjects revaccinated with DENV-3 vaccine, 1 showed a secondary response to DENV-2, while neither showed such response to DENV-3. All 4 control subjects developed dengue fever from challenge. Protection was associated with presence of NAb, although 1 subject was protected despite a lack of measurable NAb at the time of DENV-1 challenge. CONCLUSIONS: Vaccination with TDV induced variable protection against subcutaneous challenge. DENV-3 experimental challenge was associated with transient but marked elevations of transaminases.

Interference and facilitation between dengue serotypes in a tetravalent live dengue virus vaccine candidate.

BACKGROUND: Live, multivalent vaccines have historically exhibited interference in humans; live dengue virus (DENV) vaccines have proven no exception. METHODS: To characterize interactions between DENV serotypes in a tetravalent live-attenuated virus vaccine candidate, we analyzed data from a factorial clinical trial in which all combinations of high- and low-dose DENV serotypes were combined in 16 live-attenuated tetravalent vaccine formulations (N = 64) and administered to flavivirus-naive adult volunteers. Regression models considered the outcomes of reactogenicity and seroconversion, controlling for all serotype doses simultaneously. Additionally, results were compared against earlier evaluations of the same viruses administered as monovalent formulations. RESULTS: DENV-1 was immunologically dominant in both monovalent and tetravalent formulations. In tetravalent formulations, DENV-1 and DENV-2 antagonized each other, with a high dose of one decreasing seroconversion to the other. However, high-dose DENV-1 significantly increased seroconversion against 3 or more serotypes, increasing seroconversion to DENV-1, DENV-3, and DENV-4. The highest reactogenicity occurred when DENV-1 was at high dose and all others were low; reactogenicity decreased with the incorporation of other high-dose serotypes. CONCLUSIONS: Interference and facilitation occurred between serotypes in the live vaccine candidate evaluated. These analyses suggest that it may be possible to exploit facilitation to increase overall seroconversion.

Primer of COVID-19 Vaccines for the Perioperative Physician.

Dr. Emanuel Papper, the founding chairman of the Department of Anesthesiology at Columbia University, was passionate about research, training, and innovation. At an event held in his honor on March 20, 2021, experts came together to discuss the coronavirus disease 2019 (COVID-19) pandemic and its myriad challenges. Dr. Wellington Sun, MD, of Vaxcellerant LLC, an expert in infectious disease and vaccine research and development, presented a "Primer of COVID-19 vaccines for the perioperative physician." Operation Warp Speed was successful in producing multiple efficacious and safe vaccines for use in the United States and around the globe. Their development and authorization for emergency use occurred in an unprecedented timespan of <1 year. Technology such as V-SAFE has helped to accrue extensive postdevelopment safety data that will be utilized for licensure of these vaccines. The COVID-19 vaccine success is tempered by the knowledge that severe acute respiratory syndrome coronavirus 2 continues its natural selection of variants that threaten the efficacy of vaccines. Important questions remain regarding the future of the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 variants, successful vaccination strategies, and preparedness for future pandemics.

Evolution of attenuating mutations in dengue-2 strain S16803 PDK50 vaccine and comparison of growth kinetics with parent virus.

A live-attenuated dengue-2 virus strain S16803 vaccine candidate that is immunogenic and safe in humans was derived by 50 passages in primary dog kidney (PDK) cells. To identify mutations associated with attenuation of the dengue-2 PDK50 vaccine strain, we determined the nucleotide changes that arose during PDK passage of the dengue-2 virus. Thirteen mutations distinguished the PDK50 virus from low-passage parent resulting in amino acid substitutions in the premembrane (E89G), envelope (E202K, N203D), nonstructural proteins NS1 (A43T), NS2A (L181F), NS2B (I26V), and NS4B (I/T108T, L112F). In addition, the PDK50 virus contained a C to T change of nucleotide 57 in the 5' non-coding region and four silent mutations of nucleotides 591, 987, 6471, and 8907. An infectious PDK50 cDNA clone virus was produced and characterized for growth kinetics in monkey (LLC-MK(2), Vero) and mosquito (C6/36) cells. Identification of mutations in the vaccine strain and availability of an infectious clone will permit systematic analysis of the importance of individual or collective mutations on attenuation of dengue virus.

DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells.

Dengue virus is a single-stranded, enveloped RNA virus that productively infects human dendritic cells (DCs) primarily at the immature stage of their differentiation. We now find that all four serotypes of dengue use DC-SIGN (CD209), a C-type lectin, to infect dendritic cells. THP-1 cells become susceptible to dengue infection after transfection of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), or its homologue L-SIGN, whereas the infection of dendritic cells is blocked by anti-DC-SIGN antibodies and not by antibodies to other molecules on these cells. Viruses produced by dendritic cells are infectious for DC-SIGN- and L-SIGN-bearing THP-1 cells and other permissive cell lines. Therefore, DC-SIGN may be considered as a new target for designing therapies that block dengue infection.

Highly sensitive detection of dengue virus nucleic acid in samples from clinically ill patients.

Dengue virus (DENV) is a major cause of febrile illness and hemorrhagic fever in tropical and subtropical regions. Typically, patients presenting with acute dengue disease are viremic but may not have yet developed detectable titers of antibody. Therefore, early diagnosis depends mostly on detection of viral components, such as the RNA. To define the potential use of transcription-mediated amplification (TMA) DENV RNA as a diagnostic tool, we first compared its analytic sensitivity using a routine real-time reverse transcription (RT)-PCR and found that TMA is approximately 10 to 100 times more sensitive. In addition, we tested acute-phase serum samples (<5 days post-symptom onset) submitted as part of laboratory-based surveillance in Puerto Rico and determined that among patients with serologically confirmed dengue infection, TMA detected DENV RNA in almost 80% of serum specimens that were negative by the RT-PCR test used for diagnosis and in all specimens with positive RT-PCR results. We conclude that TMA is a highly sensitive method which can detect DENV RNA in approximately 89% of clinical, acute-phase serum specimens.

Phase 2 clinical trial of three formulations of tetravalent live-attenuated dengue vaccine in flavivirus-naïve adults.

Sixteen dose formulations of our live-attenuated tetravalent dengue virus vaccines (TDV) were previously evaluated for safety and immunogenicity. Two of the sixteen candidate TDV formulations (Formulations 13 and 14) were selected for further evaluation. A new TDV formulation, Formulation 17, using a higher primary dog kidney (PDK) cell passage Dengue-1 virus (DENV-1) and a lower PDK cell passage DENV-4, was developed to optimize the neutralizing antibody response. All three formulations consist of combinations of 10exp3-5 pfu/dose of the four dengue vaccine virus serotypes. This double-blind, randomized trial in 71 healthy adult subjects evaluated vaccine safety, reactogenicity and immunogenicity. TDV's were given subcutaneously in the deltoid on Day 0 and 180 (6 months). Subjects were seen in clinic on Study Days 0, 10, 28, 180, 190 and 208 and filled out daily symptom diaries for 21 days after each vaccination. Formulation 13 was the most reactogenic, while both Formulations 14 and 17 were similar in reported reactions. Seventy-five percent, 31% and 31% of subjects were viremic on Day 10 after primary vaccination with Formulations 13, 14 and 17 respectively. Viremia was not detected in any subject following the second dose of vaccine. The immunogenicity endpoint was neutralizing antibody titer one month after the second vaccination. Thirty-six percent, 40% and 63% of vaccinated subjects developed tetravalent neutralizing antibodies after two doses of Formulations 13, 14 and 17, respectively. Formulation 17 was selected for further clinical evaluation based on this study.

Safety and immunogenicity of a tetravalent live-attenuated dengue vaccine in flavivirus naive children.

A live-attenuated tetravalent dengue virus (DENV) vaccine candidate has been well tolerated and immunogenic in healthy, US flavivirus naive adult volunteers. We conducted a pilot, safety, and immunogenicity trial of the vaccine candidate in healthy Thai children (6-7 years of age) to prepare for its eventual evaluation in Thai infants. In an uncontrolled, open clinical trial, the investigational vaccine was administered on study Days 0 and 180 to seven volunteers residing in Bangkok without neutralizing antibodies to DENV1-4 or to Japanese encephalitis virus (JEV). Clinical and laboratory safety assessments were completed during the 30 days after each vaccine dose, and immunogenicity was determined at Day 30. In this study, the vaccine was well tolerated with no serious adverse events or alert laboratory values. One volunteer experienced fever (38.2 degrees C, < 2 days) and associated DENV4 vaccine viremia 7 days after Dose 2. One month after Dose 2, six volunteers in the per-protocol analysis exhibited a tetravalent neutralizing antibody response with DENV1-4 geometric mean titers of 55, 475, 350, and 171, respectively. Ten weeks (~75 days) after Dose 2, five of the six volunteers continued to exhibit a tetravalent neutralizing antibody profile; one volunteer's DENV4 PRNT50 titer fell below the assay cut-off (29 --> < 10); (clinicaltrials.gov NCT00384670).

Sonographic findings of healthy volunteers infected with dengue virus.

PURPOSE: Sonography has historically been used in developing countries to help diagnose dengue infection during epidemics of dengue hemorrhagic fever in endemic areas and to predict the clinical course. In this article, we describe the sonographic findings in subjects infected with attenuated, monovalent strains of dengue virus. METHODS: As part of a major research protocol to validate challenge strains of dengue virus for use in vaccines, 12 subjects were infected with 1 of 4 strains of dengue virus, and 3 subjects received placebo. The challenge was followed by an observation period. During this time, they were imaged regardless of the development of symptoms. RESULTS: Seven of 12 subjects infected with dengue virus showed sonographic evidence of subclinical plasma leakage, including perihepatic and perisplenic ascites, pericardial effusion, and gallbladder wall thickening. None of the 3 placebo recipients developed effusions. CONCLUSION: Sonographic evidence of fluid collection was seen in over half of subjects infected with dengue virus who did not show any evidence of dengue hemorrhagic fever. These findings shed light on possible mechanisms of plasma leakage and its role in the pathogenesis of dengue fever and dengue hemorrhagic fever.

Clinical development and regulatory points for consideration for second-generation live attenuated dengue vaccines.

Licensing and decisions on public health use of a vaccine rely on a robust clinical development program that permits a risk-benefit assessment of the product in the target population. Studies undertaken early in clinical development, as well as well-designed pivotal trials, allow for this robust characterization. In 2012, WHO published guidelines on the quality, safety and efficacy of live attenuated dengue tetravalent vaccines. Subsequently, efficacy and longer-term follow-up data have become available from two Phase 3 trials of a dengue vaccine, conducted in parallel, and the vaccine was licensed in December 2015. The findings and interpretation of the results from these trials released both before and after licensure have highlighted key complexities for tetravalent dengue vaccines, including concerns vaccination could increase the incidence of dengue disease in certain subpopulations. This report summarizes clinical and regulatory points for consideration that may guide vaccine developers on some aspects of trial design and facilitate regulatory review to enable broader public health recommendations for second-generation dengue vaccines.

FDA licensure of and ACIP recommendations for vaccines.

Many healthcare providers are not familiar with the Food and Drug Administration (FDA) vaccine licensure process, the Advisory Committee on Immunization Practices (ACIP) vaccine recommendation process, and how FDA vaccine licensure and ACIP recommendations are related. Vaccines for use in the United States military and civilian populations are licensed by the FDA by several potential pathways but use of licensed vaccines in the civilian population should be based on recommendations made by the ACIP. In performing these distinct activities, FDA and ACIP function under different mandates. In this article, we discuss whether the FDA licensure pathways used to approve a vaccine impacts ACIP recommendation categories for vaccines licensed from 2006 to 2016.

Emergence of a new human adenovirus type 4 (Ad4) genotype: identification of a novel inverted terminal repeated (ITR) sequence from majority of Ad4 isolates from US military recruits.

BACKGROUND: Ad4 is the principal etiological agent of acute respiratory disease (ARD) in the US military. Discovery of the novel 208bp inverted terminal repeated (ITR) sequence from a recent Ad4 Jax78 field isolate was totally distinct from the analogous 116bp ITR of Ad4 prototype. OBJECTIVES: To investigate the origin and distribution of the novel Ad4 ITR sequence from ARD infections. STUDY DESIGN: Direct sequencing of ligated Ad ITR termini. RESULTS: The new Ad4 ITR was highly homologous with the ITRs of human Ad subgroup B. The left post-ITR region of Ad4 Jax78 was found to be highly homologous to the corresponding region of subgroup B Ads: 81% for Ad11 and 98% for Ad3 and Ad7. The right post-ITR region of Ad4 Jax78 contained a truncated classic ITR of the Ad4 prototype. CONCLUSIONS: The Ad4 Jax78 ITR most likely evolved from Ad4 prototype by substituting the Ad4 prototype ITR with the subgroup B Ads ITR. The ITR-based PCR assays developed from this study can be used to distinguish the new Ad4 genotype from the classical Ad4 prototype. The new Ad4 genotype was first detected in 1976 from Georgia, USA, and is the main causative agent of ARD infections in US military population.

An immunocytometric assay based on dengue infection via DC-SIGN permits rapid measurement of anti-dengue neutralizing antibodies.

Dengue remains a global public health threat and development of a safe and effective vaccine is a principal public health goal. The primary correlate of immunity is thought to be neutralizing antibodies. Currently, the plaque reduction neutralization test (PRNT) is the gold standard measure of dengue neutralizing antibody responses, but this test is limited by time-consuming performance. In addition, some feel that use of viral strains adapted to grow in Vero or BHK cells may not accurately reflect protective responses. A human cell line transfected to express a putative natural dengue receptor, DC-SIGN (CD209), was used to measure antibody-mediated dengue neutralization. Using neutralizing monoclonal antibodies, immune sera, and laboratory adapted dengue viruses, serotype-specific neutralizing activity was demonstrated similar to that seen in the Vero PRNT. Importantly, serotype-specific neutralizing activity against recently isolated dengue strains with less heterotypic cross-neutralization than laboratory adapted viruses was also demonstrated.

Human dendritic cells and macrophages exhibit different intracellular processing pathways for soluble and liposome-encapsulated antigens.

The intracellular fates of soluble and liposomal antigens in human macrophages and dendritic cells are not well defined. Previous studies using murine macrophages have demonstrated that liposomal antigens can enter the MHC class I pathway. The Golgi complex is a major organelle in this pathway. Phagocytosis of the antigens is followed by translocation of antigen-derived peptides to the trans-Golgi where they can complex with MHC class I molecules. In contrast, soluble antigens are normally processed through the MHC class II pathway. Therefore, in the present study, ovalbumin and a synthetic Ebola peptide were used either in a soluble form or encapsulated in liposomes to investigate the intracellular trafficking and localization of these antigens to the Golgi complex in human macrophages and dendritic cells. While liposome-encapsulated antigens were transported to the trans-Golgi region in 59-78% of macrophages, soluble antigens remained diffuse throughout the cytoplasm with only 3-11% of the macrophages exhibiting trans-Golgi localization. The majority of dendritic cells localized both soluble (Ebola, 75%; ovalbumin, 84%) and liposomal antigens (58% and 65%), and irradiated Ebola virus to the trans-Golgi. These studies demonstrate that the intracellular fate of soluble and liposomal antigens can differ depending upon the antigen-presenting cell.

Incidence of dengue virus infection in school-aged children in Puerto Rico: a prospective seroepidemiologic study.

Dengue is a potentially fatal acute febrile illness caused by the mosquito-borne dengue viruses (DENV-1 to -4). To estimate DENV seroincidence in school-aged children, a 1-year prospective cohort study was conducted in Patillas, Puerto Rico; 10- to 18-year-olds (N = 345) were randomly selected from 13 public schools. At enrollment, 49.8% of the entire cohort had DENV immunoglobulin G (IgG) anti-DENV antibodies, and there were individuals with neutralizing antibodies specific to each of the four DENV. The mean age of participants with incident DENV infection was 13.4 years. The 1-year seroincidence rate was 5.6%, and 61.1% of infections were inapparent. Having IgG anti-DENV at enrollment was associated with seroincidence (risk ratio = 6.8). Acute febrile illnesses during the study period were captured by a fever diary and an enhanced and passive surveillance system in the municipios of Patillas and Guayama. In summary, at enrollment, nearly one-half of the participants had a prior DENV infection, with the highest incidence in the 10- to 11-year-olds, of which most were inapparent infections, and symptomatic infections were considered mild.

A Phase II, Randomized, Safety and Immunogenicity Trial of a Re-Derived, Live-Attenuated Dengue Virus Vaccine in Healthy Children and Adults Living in Puerto Rico.

This was a double-blind, randomized, controlled, phase II clinical trial, two dose study of re-derived, live-attenuated, tetravalent dengue virus (TDEN) vaccine (two formulations) or placebo in subjects 1-50 years of age. Among the 636 subjects enrolled, 331 (52%) were primed, that is, baseline seropositive to at least one dengue virus (DENV) type. Baseline seropositivity prevalence increased with age (10% [< 2 years], 26% [2-4 years], 60% [5-20 years], and 93% [21-50 years]). Safety profiles of TDEN vaccines were similar to placebo regardless of priming status. No vaccine-related serious adverse events (SAEs) were reported. Among unprimed subjects, immunogenicity (geometric mean antibody titers [GMT] and seropositivity rates) for each DENV increased substantially in both TDEN vaccine groups with at least 74.6% seropositive for four DENV types. The TDEN vaccine candidate showed an acceptable safety and immunogenicity profile in children and adults ranging from 1 to 50 years of age, regardless of priming status. ClinicalTrials.gov: NCT00468858.