Development of recombinant rotavirus carrying herpes simplex virus 2 glycoprotein D gene based on reverse genetics technology.

Vaccine development for herpes simplex virus 2 (HSV-2) has been attempted, but no vaccines are yet available. A plasmid-based reverse genetics system for Rotavirus (RV), which can cause gastroenteritis, allows the generation of recombinant RV containing foreign genes. In this study, we sought to develop simian RV (SA11) as a vector to express HSV-2 glycoprotein D (gD2) and evaluated its immunogenicity in mice. We generated the recombinant SA11-gD2 virus (rSA11-gD2) and confirmed its ability to express gD2 in vitro. The virus was orally inoculated into suckling BALB/c mice and into 8-week-old mice. Serum IgG and IgA titers against RV and gD2 were measured by ELISA. In the 8-week-old mice inoculated with rSA11-gD2, significant increases in not only antibodies against RV but also IgG against gD2 were demonstrated. In the suckling mice, antibodies against RV were induced, but gD2 antibody was not detected. Diarrhea observed after the first inoculation of rSA11-gD2 in suckling mice was similar to that induced by the parent virus. A gD2 expressing simian RV recombinant, which was orally inoculated, induced IgG against gD2. This strategy holds possibility for genital herpes vaccine development.

Approaches to demonstrating the effectiveness of filovirus vaccines: Lessons from Ebola and COVID-19.

Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV) and Marburg virus (MARV), are members of the Filoviridae family that can cause severe disease and death in humans and animals. The reemergence of Ebola, Sudan and Marburg virus disease highlight the need for continued availability of safe and effectives vaccines as well as development of new vaccines. While randomized controlled trials using disease endpoints provide the most robust assessment of vaccine effectiveness, challenges to this approach include the unpredictable size, location, occurrence and duration of filovirus disease outbreaks. Thus, other approaches to demonstrating vaccine effectiveness have been considered. These approaches are discussed using examples of preventive vaccines against other infectious diseases. In addition, this article proposes a clinical immunobridging strategy using licensed EBOV vaccines as comparators for demonstrating the effectiveness of filovirus vaccine candidates that are based on the same licensed vaccine platform technology.

Reliably Assessing Duration of Protection for Coronavirus Disease 2019 Vaccines.

Decision making about vaccination and boosting schedules for coronavirus disease 2019 (COVID-19) hinges on reliable methods for evaluating the longevity of vaccine protection. We show that modeling of protection as a piecewise linear function of time since vaccination for the log hazard ratio of the vaccine effect provides more reliable estimates of vaccine effectiveness at the end of an observation period and also detects plateaus in protective effectiveness more reliably than the standard method of estimating a constant vaccine effect over each time period. This approach will be useful for analyzing data pertaining to COVID-19 vaccines and other vaccines for which rapid and reliable understanding of vaccine effectiveness over time is desired.

SARS-CoV-2 Variants and Vaccines.

Viral variants of concern may emerge with dangerous resistance to the immunity generated by the current vaccines to prevent coronavirus disease 2019 (Covid-19). Moreover, if some variants of concern have increased transmissibility or virulence, the importance of efficient public health measures and vaccination programs will increase. The global response must be both timely and science based.

COVID-19 vaccine trials: The potential for "hybrid" analyses.

BACKGROUND: Although several COVID-19 vaccines have been found to be effective in rigorous evaluation and have emerging availability in parts of the world, their supply will be inadequate to meet international needs for a considerable period of time. There also will be continued interest in vaccines that are more effective or have improved scalability to facilitate mass vaccination campaigns. Ongoing clinical testing of new vaccines also will be needed as variant strains continue to emerge that may elude some aspects of immunity induced by current vaccines. Randomized clinical trials meaningfully enhance the efficiency and reliability of such clinical testing. In clinical settings with limited or no access to known effective vaccines, placebo-controlled randomized trials of new vaccines remain a preferred approach to maximize the reliability, efficiency and interpretability of results. When emerging availability of licensed vaccines makes it no longer possible to use a placebo control, randomized active comparator non-inferiority trials may enable reliable insights. METHODS: In this article, "hybrid" methods are proposed to address settings where, during the conduct of a placebo-controlled trial, a judgment is made to replace the placebo arm by a licensed COVID-19 vaccine due to emerging availability of effective vaccines in regions participating in that trial. These hybrid methods are based on proposed statistics that aggregate evidence to formally test as well as to estimate the efficacy of the experimental vaccine, by combining placebo-controlled data during the first period of trial conduct with active-controlled data during the second period. RESULTS: Application of the proposed methods is illustrated in two important scenarios where the active control vaccine would become available in regions engaging in the experimental vaccine's placebo-controlled trial: in the first, the active comparator's vaccine efficacy would have been established to be 50%-70% for the 4- to 6-month duration of follow-up of its placebo-controlled trial; in the second, the active comparator's vaccine efficacy would have been established to be 90%-95% during that duration. These two scenarios approximate what has been seen with adenovirus vaccines or mRNA vaccines, respectively, assuming the early estimates of vaccine efficacy for those vaccines would hold over longer-term follow-up. CONCLUSION: The proposed hybrid methods could readily play an important role in the near future in the design, conduct and analysis of randomized clinical trials performed to address the need for multiple additional vaccines reliably established to be safe and have worthwhile efficacy in reducing the risk of symptomatic disease from SARS-CoV-2 infections.

COVID-19 vaccine trials: The use of active controls and non-inferiority studies.

BACKGROUND: Recently emerging results from a few placebo-controlled randomized trials of COVID-19 vaccines revealed estimates of 62%-95% relative reductions in risk of virologically confirmed symptomatic COVID-19 disease, over approximately 2-month average follow-up period. Additional safe and effective COVID-19 vaccines are needed in a timely manner to adequately address the pandemic on an international scale. Such safe and effective vaccines would be especially appealing for international deployment if they also have favorable stability, supply, and potential for implementation in mass vaccination campaigns. Randomized trials provide particularly reliable insights about vaccine efficacy and safety. While enhanced efficiency and interpretability can be obtained from placebo-controlled trials, in settings where their conduct is no longer possible, randomized non-inferiority trials may enable obtaining reliable evaluations of experimental vaccines through direct comparison with active comparator vaccines established to have worthwhile efficacy. METHODS: The usual objective of non-inferiority trials is to reliably assess whether the efficacy of an experimental vaccine is not unacceptably worse than that of an active control vaccine previously established to be effective, likely in a placebo-controlled trial. This is formally achieved by ruling out a non-inferiority margin identified to be the minimum threshold for what would constitute an unacceptable loss of efficacy. This article not only investigates non-inferiority margins, denoted by δ, that address the usual objective of determining whether the experimental vaccine is "at least similarly effective to" the active comparator vaccine in the non-inferiority trial, but also develops non-inferiority margins, denoted by δo, intended to address the worldwide need for multiple safe and effective vaccines by satisfying the less stringent requirement that the experimental vaccine be "at least similarly effective to" an active comparator vaccine having efficacy that satisfies the widely accepted World Health Organization-Food and Drug Administration criteria for "worthwhile" vaccine efficacy. RESULTS: Using the margin δ enables non-inferiority trials to reliably evaluate experimental vaccines that truly are similarly effective to an active comparator vaccine having any level of "worthwhile" efficacy. When active comparator vaccines have efficacy in the range of 50%-70%, non-inferiority trials designed to use the margin δo have appealing properties, especially for experimental vaccines having true efficacy of approximately 60%. CONCLUSION: Non-inferiority trials using the proposed margins may enable reliable randomized evaluations of efficacy and safety of experimental COVID-19 vaccines. Such trials often require approximately two- to three-fold the person-years follow-up than a placebo-controlled trial. This could be achieved, without substantive increases in sample size, by increasing the average duration of follow-up from 2 months to approximately 4-6 months, assuming efficacy of the active comparator vaccine has been reliably evaluated over that longer duration.

Viewpoint of a WHO Advisory Group Tasked to Consider Establishing a Closely-monitored Challenge Model of Coronavirus Disease 2019 (COVID-19) in Healthy Volunteers.

WHO convened an Advisory Group (AG) to consider the feasibility, potential value, and limitations of establishing a closely-monitored challenge model of experimental severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) in healthy adult volunteers. The AG included experts in design, establishment, and performance of challenges. This report summarizes issues that render a COVID-19 model daunting to establish (the potential of SARS-CoV-2 to cause severe/fatal illness, its high transmissibility, and lack of a "rescue treatment" to prevent progression from mild/moderate to severe clinical illness) and it proffers prudent strategies for stepwise model development, challenge virus selection, guidelines for manufacturing challenge doses, and ways to contain SARS-CoV-2 and prevent transmission to household/community contacts. A COVID-19 model could demonstrate protection against virus shedding and/or illness induced by prior SARS-CoV-2 challenge or vaccination. A limitation of the model is that vaccine efficacy in experimentally challenged healthy young adults cannot per se be extrapolated to predict efficacy in elderly/high-risk adults.

Animal models for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.

Scientific and Regulatory Considerations for Efficacy Studies of Cytomegalovirus Vaccines.

The considerable public health burden due to cytomegalovirus (CMV) supports current interest in vaccine development. Clinical studies intended to support regulatory action should be designed to demonstrate substantial evidence of effectiveness. However, design and conduct of clinical endpoint studies may be hampered by low incidence of disease, especially for congenital CMV. Discussion and experience from other vaccines directed against congenital disease (including rubella and Zika) may be instructive. This article summarizes current scientific and US regulatory considerations related to design of studies of vaccines intended to prevent congenital CMV and complications of CMV in transplantation, as discussed at the 2018 workshop entitled "Cytomegalovirus Infection: Advancing Strategies for Prevention and Treatment."

Hidden regulation of herpes simplex virus 1 pre-mRNA splicing and polyadenylation by virally encoded immediate early gene ICP27.

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.

Herpes Simplex Virus 2 in Autonomic Ganglia: Evidence for Spontaneous Reactivation.

Herpes simplex virus 2 (HSV-2) can be transmitted in the presence or absence of lesions, allowing efficient spread among the general population. Recurrent HSV genital lesions are thought to arise from reactivated latent virus in sensory cell bodies of the dorsal root ganglia (DRG). However, HSV-2 has also been found latent in autonomic ganglia. Spontaneous reactivation or a low level of chronic infection could theoretically also occur in these peripheral nervous tissues, contributing to the presence of infectious virus in the periphery and to viral transmission. Use of a recently described, optimized virus with a monomeric mNeonGreen protein fused to viral capsid protein 26 (VP26) permitted detection of reactivating virus in explanted ganglia and cryosections of DRG and the sacral sympathetic ganglia (SSG) from latently infected guinea pigs. Immediate early, early, and late gene expression were quantified by droplet digital reverse transcription-PCR (ddRT-PCR), providing further evidence of viral reactivation not only in the expected DRG but also in the sympathetic SSG. These findings indicate that viral reactivation from autonomic ganglia is a feature of latent viral infection and that these reactivations likely contribute to viral pathogenesis.IMPORTANCE HSV-2 is a ubiquitous important human pathogen that causes recurrent infections for the life of its host. We hypothesized that the autonomic ganglia have important roles in viral reactivation, and this study sought to determine whether this is correct in the clinically relevant guinea pig vaginal infection model. Our findings indicate that sympathetic ganglia are sources of reactivating virus, helping explain how the virus causes lifelong recurrent disease.

Towards dynamic monitoring of cell cultures using high throughput sequencing.

We used a combination of DOP-PCR with high throughput sequencing (HTS) to study infected cell cultures over time to assess the feasibility of using this technique to provide a read-out other than cytopathic effect in cell culture infectivity assays. Because DOP-PCR primers feature a short constant sequence at their 3' terminus, the procedure yields a reproducible representational library of products from a given PCR template, including viral nucleic acids. Using SV40- and MVM-infected cultures harvested at different times, we show that the number of viral matches among DOP-PCR products parallels the quantity of virus as shown by real-time PCR, and further show that HTS analysis of specific DOP-PCR products that increase in quantity over time could be used to identify the infecting virus with a sensitivity similar to that of typical cell-culture assays that rely on cytopathic effect.

Demonstrating vaccine effectiveness during a waning epidemic: A WHO/NIH meeting report on approaches to development and licensure of Zika vaccine candidates.

Since its peak in early 2016, the incidence of Zika virus (ZIKV) cases has declined to such low levels that Phase 3 field efficacy trials may be infeasible. While great progress was made to rapidly advance several vaccine candidates into Phase 1 and 2 clinical trials, in the absence of sustained viral transmission it may be difficult to evaluate the effectiveness of ZIKV vaccine candidates by conducting traditional clinical disease endpoint efficacy studies. However, ZIKV is still circulating at low levels in some areas and is likely to re-emerge in naïve populations or in sites of prior epidemics once population immunity wanes. Therefore, the public health need for a ZIKV vaccine remains. To facilitate continued ZIKV vaccine development efforts, the World Health Organization's Initiative for Vaccine Research and the National Institutes of Health's National Institute of Allergy and Infectious Diseases co-hosted a meeting of experts in March 2018 to identify strategies to demonstrate vaccine effectiveness in view of waning ZIKV disease incidence. This paper outlines points for consideration for developers, regulators, and other stakeholders working towards a licensed ZIKV vaccine. These deliberations may also be applicable to development of vaccines for other emerging infections where the size, unpredictability, and ephemeral nature of outbreaks makes clinical disease endpoint efficacy trials to demonstrate vaccine effectiveness infeasible.