Biology subjects

Brien, J. D.

Publications and source records attributed to Brien, J. D..

10 recordsLinked to original sources

bioRxiv2025

mRNA-LNP Vaccines Encoding for Dengue Optimized prM/ENV Proteins Induce Protective Immunity without ADE

Dengue virus (DENV) is the most common mosquito-borne virus in the world, causing nearly 400 million infections annually, with the number of cases predict to increase over time. Despite this prevalence there are no widely approved vaccine for DENV naive individuals or therapeutics. DENV consists of four distinct serotypes, DENV 1-4, that share 60-70% homology. Development of a safe and efficacious pan-Dengue vaccine has been complicated by the potential of antibody-dependent enhancement, in which cross-reactive non-neutralizing antibodies can enhance infection and disease. We previously demonstrated that a mRNA-LNP encoding for DENV-1 prM and Envelope proteins generates homotypic neutralizing immune responses and protects against a lethal challenge. The DENV-1 vaccine avoided ADE by eliminating the fusion loop (FL), one of the dominant ADE epitopes. Using a similar design methodology, here we present mRNA-LNP prM-E vaccines for DENV 2, 3, and 4. These vaccines maintained the {Delta}FL modifications, but encode a chimeric E protein to improve VLP expression and stability. All three vaccines elicited neutralizing titers of serotype specific antibodies mice and protected against a lethal homotypic DENV challenge. Mutation of the FL lowered ADE in vaccinated mouse sera. We formulated monotypic vaccines into a tetravalent vaccine and evaluated for immunogenicity and protection in mice. The tetravalent vaccine elicited neutralizing humoral responses against all DENV serotypes and protected against lethal challenges. Cumulatively, our monovalent DENV mRNA-LNP vaccines against all DENV serotypes generate protective immunity and lower the potential for ADE, leading to the development of a first-generation ADE-altered tetravalent DENV vaccine.

microbiology↗

bioRxiv2024

The TLR7/8 agonist INI-4001 enhances the immunogenicity of a Powassan virus-like-particle vaccine

Powassan virus (POWV) is a pathogenic tick-borne flavivirus that causes fatal neuroinvasive disease in humans. There are currently no approved therapies or vaccines for POWV infection. Here, we develop a POW virus-like-particle (POW-VLP) based vaccine adjuvanted with the novel synthetic Toll-like receptor 7/8 agonist INI-4001. We demonstrate that INI-4001 outperforms both alum and the Toll-like receptor 4 agonist INI-2002 in enhancing the immunogenicity of a dose-sparing POW-VLP vaccine in mice. INI-4001 increases the magnitude and breadth of the antibody response as measured by whole-virus ELISA, induces neutralizing antibodies measured by FRNT, reduces viral burden in the brain of infected mice measured by RT qPCR, and confers 100% protection from lethal challenge with both lineages of POWV. We show that the antibody response induced by INI-4001 is more durable than standard alum, and 80% of mice remain protected from lethal challenge 9-months post-vaccination. Lastly, we show that the protection elicited by INI-4001 adjuvanted POW-VLP vaccine is unaffected by either CD4+ or CD8+ T cell depletion and can be passively transferred to unvaccinated mice indicating that protection is mediated through humoral immunity. This study highlights the utility of novel synthetic adjuvants in VLP-based vaccines. Author summaryPowassan virus (POWV) is an emerging pathogenic tick-borne flavivirus for which there is no vaccine. Current tick-borne flavivirus vaccines are less than ideal and use formalin-inactivated virus adjuvanted with alum. These vaccines require thorough inactivation of the antigen and frequent boosting to maintain immunity. In this study, we describe the development of a POWV vaccine using Powassan virus-like-particles (POW-VLPs) adjuvanted with either of two novel Toll-like receptor (TLR) agonists, the TLR4 agonist INI-2002 or the TLR7/8 agonist INI-4001. We show that INI-4001 enhances the antibody response, reduces POWV neuroinvasion, and elicits full protection from lethal POWV infection in mice prime-boost vaccinated with low doses of POW-VLP. We further show that this protection is mediated by a humoral immune response which is both broader and more durable than a POW-VLP vaccine formulated with alum. These findings demonstrate the effectiveness of the novel synthetic TLR7/8 agonist INI-4001 as an adjuvant for low-dose VLP-based vaccines and the ability of this vaccine platform to improve upon current tick-borne flavivirus vaccine methodology.

immunology↗

bioRxiv2021

A mRNA-LNP vaccine against Dengue Virus elicits robust, serotype-specific immunity

Dengue virus (DENV) is the most common vector-borne viral disease with nearly 400 million worldwide infections each year concentrated in the tropical and subtropical regions of the world. Severe dengue complications are often associated with a secondary heterotypic infection of one of the four circulating serotypes. In this scenario, humoral immune responses targeting cross-reactive, poorly-neutralizing epitopes can lead to increased infectivity of susceptible cells via antibody-dependent enhancement (ADE). In this way, antibodies produced in response to infection or vaccination are capable of contributing to enhanced disease in subsequent infections. Currently, there are no available therapeutics to combat DENV disease, and there is an urgent need for a safe and efficacious vaccine. Here, we developed a nucleotide-modified mRNA vaccine encoding for the membrane and envelope structural proteins from DENV serotype 1 encapsulated into lipid nanoparticles (prM/E mRNA-LNP). Vaccination of mice elicited robust antiviral immune responses comparable to viral infection with high levels of neutralizing antibody titers and antiviral CD4+ and CD8+ T cells. Immunocompromised AG129 mice vaccinated with the prM/E mRNA-LNP vaccine were protected from a lethal DENV challenge. Vaccination with either a wild-type vaccine, or a vaccine with mutations in the immunodominant fusion-loop epitope, elicited equivalent humoral and cell mediated immune responses. Neutralizing antibodies elicited by the vaccine were sufficient to protect against a lethal challenge. Both vaccine constructs demonstrated serotype specific immunity with minimal serum cross-reactivity and reduced ADE compared to a live DENV1 viral infection. IMPORTANCEWith 400 million worldwide infections each year, dengue is the most common vector-born viral disease. 40% of the worlds population is at risk with dengue experiencing consistent geographic spread over the years. With no therapeutics available and vaccines performing sub optimally, the need for an effective dengue vaccine is urgent. Here we develop and characterize a novel mRNA vaccine encoding for the dengue serotype 1 envelope and premembrane structural proteins that is delivered via a lipid nanoparticle. Our DENV1 prM/E mRNA-LNP vaccine induces neutralizing antibody and cellular immune responses in immunocompetent mice and protects an immunocompromised mouse from a lethal DENV challenge. Existing antibodies against dengue can enhance subsequent infections via antibody-dependent enhancement. Importantly our vaccine only induced serotype specific immune responses and did not induce ADE.

microbiology↗

bioRxiv2020

Heterologous flavivirus exposure provides varying degrees of cross-protection from Zika virus in a mouse model of infection

The 2015/16 Zika virus epidemic in South and Central America left the scientific community urgently trying to understand the disease and the factors which modulate Zika virus pathogenesis. Multiple other flaviviruses are endemic in areas where Zika virus emerged in 2015/16. Therefore, it is hypothesized that a key to understanding how Zika virus infection and disease progresses, is to study Zika virus infection in the context of prior flavivirus exposure. Humans and animal studies have highlighted the idea that having been previously exposed to a heterologous flavivirus may modulate the immune response to Zika virus. However, it is still unclear 1) how this impacts viral burden and pathology, and 2) the factors which correlate with the multiple metrics of disease. In this murine study, we longitudinally examine multiple factors involved in Zika disease, linking viral burden over time with increased neurological disease severity and weight loss. We show that prior heterologous flavivirus exposure with dengue virus type 2 or 3, or the vaccine strain of yellow fever, provides protection from mortality in a lethal Zika challenge. Reduction in viral burden and Zika disease in the context of prior flavivirus exposure varies depending on the infecting primary virus; with primary Zika infection being most protective from Zika challenge, followed by dengue 2, yellow fever, and dengue 3. This study demonstrates a protective effect of prior heterologous flavivirus exposure on Zika virus pathogenesis, and defines the relationship between prior flavivirus exposure and the potential for Zika virus disease. IMPORTANCEThe emergence and re-emergence of various vector-borne diseases in recent years highlights the need to understand the mechanisms of protection for each pathogen. In this study, we investigated the impact of prior exposure to Zika, dengue serotypes 2, 3, and the vaccine strain of yellow fever on pathogenesis and disease outcomes in a mouse model of Zika virus infection. We found that prior exposure to a heterologous flavivirus was protective from mortality, neurological disease, weight loss, and severe viral burden during a lethal Zika challenge. Using a longitudinal study design, we were able to link multiple disease parameters including viral burden over time with neurological disease severity and weight loss in the context of heterologous infection. This study demonstrates a role for heterologous flavivirus exposure in modulating flavivirus pathophysiology. Given the cyclic nature of most flavivirus outbreaks, this work will contribute to the forecasting of disease severity for future outbreaks.

microbiology↗

bioRxiv2020

The receptor binding domain of SARS-CoV-2 spike is the key target of neutralizing antibody in human polyclonal sera.

Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. These data provide critical information for vaccine development and development of sensitive and specific serological testing.

immunology↗

bioRxiv2020

Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

pathology↗

bioRxiv2020

mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in nearly 20 million infections across the globe, as of August 2020. Critical to the rapid evaluation of vaccines and antivirals is the development of tractable animal models of infection. The use of common laboratory strains of mice to this end is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Interestingly, we did not observe an enhancement of SARS-CoV-2 specific antibody responses with hACE2 induction. Importantly, using this system, we functionally identified the CD4+ and CD8+ peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice. Antigen-specific CD8+ T cells in mice of this MHC haplotype primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. The functional identification of these T cell epitopes will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2. The use of this tractable expression system has the potential to be used in other instances of emerging infections in which the rapid development of an animal model is hindered by a lack of host susceptibility factors.

immunology↗

bioRxiv2020

T cell responses induced by attenuated flavivirus vaccination are specific and show limited cross-reactivity with other flavivirus species.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. Here, we tested pools of epitopes derived from dengue (DENV), zika (ZIKV), Japanese Encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by Intracellular Cytokine Staining (ICS) using PBMCs of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccines. CD8 T cell responses recognized epitopes from multiple flaviviruses, however, the magnitude of cross-reactive responses was consistently several-fold lower than those to the autologous epitope pools, and associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing twenty-nine different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with more than 100-fold lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses, and in five out of eight cases more than 1000-fold lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and has implications for understanding immunity elicited by natural infection, and strategies to develop live attenuated vaccines against flaviviral species. ImportanceThe envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the non-structural (NS) and capsid (C) antigens which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses amongst different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses, and might thus impair vaccine performance.

immunology↗

bioRxiv2019

Significant control of Zika infection in macaques depends on the elapsing time after dengue exposure

Prior exposure to a single serotype of dengue virus (DENV) predisposes individuals to severe disease upon secondary heterologous DENV infection. Here we show that the length of time between DENV/Zika (ZIKV) infections has a qualitative impact on controlling ZIKV replication. We identified limited but significant differences in the magnitude of the early humoral immune response associated with a period of twelve months but not three months of DENV convalescence. However, their role limiting ZIKV replication is not conclusive. There was no evidence of in vivo antibody-dependent amplification of ZIKV by DENV immunity in any group. We are also showing that the significant differences among groups may be linked to a pre-existing polyfunctional CD4+ T cells response (increased IFN-g and Cd107a before ZIKV infection) and to an early and continuous expansion of the CD4+ effector memory cells early on after ZIKV infection. Those significant differences were associated with a period of 12 months after DENV infection that were not observed in a span of 3-months. These results suggest that there is a window of optimal cross-protection between ZIKV and DENV with significant consequences. These results have pivotal implications while interpreting ZIKV pathogenesis in flavivirus-experimented populations, diagnostic results interpretation and vaccine designs among others.\n\nAuthor SummarySince its introduction in the Americas region ZIKV virus has been associated to severe birth defects. One of the questions that remains open is the role of previous dengue or any other flavivirus immunity in the pathogenesis of ZIKV and more important, if the time elapse between DENV and ZIKV play a role enhancing ZIKV pathogenesis as it is the case for subsequent DENV infections. On this work, using NHP as a model we compared the effect of a period of 12 months vs. a period of 3 months of DENV immunity in the outcome of ZIKV infection. We found that previous DENV infection, at any of the tested period of time do not induce ZIKV enhancement. More relevant are showing that when the two infection occurs at least one year apart the preexisting DENV immunity is better at controlling ZIKV replication and that the role of the neutralizing antibodies is very limited. On the contrary our results suggest that early after ZIKV infection the cellular immune response, may plays a predominant role. Our findings have critical relevance to understand the dynamic interaction between these two flavivirus, their pathogenies, diagnosis and vaccine design.

immunology↗

bioRxiv2019

Time elapsed between Zika and Dengue infections improves the immune response against Dengue without viremia enhancement in rhesus macaques

The role of Zika virus (ZIKV) immunity on subsequent dengue virus (DENV) infections is relevant to anticipate the dynamics of forthcoming DENV epidemics in areas with previous ZIKV exposure. We study the effect of ZIKV infection with various strains on subsequent DENV immune response after 10 and 2 months of ZIKV infection in rhesus macaques. Our results show that a subsequent DENV infection in animals with early- and middle-convalescent periods to ZIKV do not promote an increase in DENV viremia nor pro-inflammatory status. Previous ZIKV exposure increases the magnitude of the antibody and T cell responses against DENV, and different time intervals between infections alter the magnitude and durability of such responses--more after longer ZIKV pre-exposure. Collectively, we find no evidence of a detrimental effect of ZIKV immunity in a subsequent DENV infection. This supports the implementation of ZIKV vaccines that could also boost immunity against future DENV epidemics.

microbiology↗