Alzheimer's disease and multiple sclerosis: a possible connection through the viral demyelinating neurodegenerative trigger (vDENT).

Alzheimer's disease (AD) and multiple sclerosis (MS) are two CNS disorders affecting millions of people, for which no cure is available. AD is usually diagnosed in individuals age 65 and older and manifests with accumulation of beta amyloid in the brain. MS, a demyelinating disorder, is most commonly diagnosed in its relapsing-remitting (RRMS) form in young adults (age 20-40). The lack of success in a number of recent clinical trials of immune- or amyloid-targeting therapeutics emphasizes our incomplete understanding of their etiology and pathogenesis. Evidence is accumulating that infectious agents such as viruses may contribute either directly or indirectly. With the emerging recognition that demyelination plays a role in risk and progression of AD, we propose that MS and AD are connected by sharing a common environmental factor (a viral infection such as HSV-1) and pathology (demyelination). In the viral DEmyelinating Neurodegenerative Trigger (vDENT) model of AD and MS, the initial demyelinating viral (e.g., HSV-1) infection provokes the first episode of demyelination that occurs early in life, with subsequent virus reactivations/demyelination and associated immune/inflammatory attacks resulting in RRMS. The accumulating damage and/or virus progression deeper into CNS leads to amyloid dysfunction, which, combined with the inherent age-related defects in remyelination, propensity for autoimmunity, and increased blood-brain barrier permeability, leads to the development of AD dementia later in life. Preventing or diminishing vDENT event(s) early in life, thus, may have a dual benefit of slowing down the progression of MS and reducing incidence of AD at an older age.

Development of Respiratory Syncytial Virus Vaccine Candidates for the Elderly.

Respiratory syncytial virus (RSV) is a significant threat to elderly populations and repeated infections that occur throughout life are poorly protective. To assess the role of prior RSV infections as well as elderly immune senescence on vaccine efficacy, we compared immune responses after virus-like particle (VLP) immunization of elderly cotton rats and young cotton rats, both previously RSV infected, in order to mimic the human population. We show that immunization of RSV-experienced young or elderly animals resulted in the same levels of anti-pre-F IgG, anti-G IgG, neutralizing antibody titers, and protection from challenge indicating that the delivery of F and G proteins in a VLP is equally effective in activation of protective responses in both elderly and young populations. Our results suggest that F and G protein-containing VLPs induce anti-RSV memory established in prior RSV infections equally well in both young and elderly animals and thus can be an effective vaccine for the elderly.

Novel neuroendocrine role of γ-aminobutyric acid and gastrin-releasing peptide in the host response to influenza infection.

Gastrin-releasing peptide (GRP), an evolutionarily conserved neuropeptide, significantly contributes to influenza-induced lethality and inflammation in rodent models. Because GRP is produced by pulmonary neuroendocrine cells (PNECs) in response to γ-aminobutyric acid (GABA), we hypothesized that influenza infection promotes GABA release from PNECs that activate GABAB receptors on PNECs to secrete GRP. Oxidative stress was increased in the lungs of influenza A/PR/8/34 (PR8)-infected mice, as well as serum glutamate decarboxylase 1, the enzyme that converts L-glutamic acid into GABA. The therapeutic administration of saclofen, a GABAB receptor antagonist, protected PR8-infected mice, reduced lung proinflammatory gene expression of C-C chemokine receptor type 2 (Ccr2), cluster of differentiation 68 (Cd68), and Toll like receptor 4 (Tlr4) and decreased the levels of GRP and high-mobility group box 1 (HMGB1) in sera. Conversely, baclofen, a GABAB receptor agonist, significantly increased the lethality and inflammatory responses. The GRP antagonist, NSC77427, as well as the GABAB antagonist, saclofen, blunted the PR8-induced monocyte infiltration into the lung. Together, these data provide the first report of neuroregulatory control of influenza-induced disease.

Human Metapneumovirus (hMPV) Infection and MPV467 Treatment in Immunocompromised Cotton Rats Sigmodon hispidus.

Human metapneumovirus (hMPV) is an important cause of respiratory disease in immunocompromised individuals, yet hMPV infection has not been modeled before in immunocompromised animals. In this work, cotton rats S. hispidus immunosuppressed by cyclophosphamide were infected with hMPV, and viral replication and pulmonary inflammation in these animals were compared to those in normal hMPV-infected S. hispidus. The efficacy of prophylactic and therapeutic administration of the anti-hMPV antibody MPV467 was also evaluated. Immunosuppressed animals had higher pulmonary and nasal titers of hMPV on day 5 post-infection compared to normal animals, and large amounts of hMPV were still present in the respiratory tract of immunosuppressed animals on days 7 and 9 post-infection, indicating prolonged viral replication. Immunosuppression was accompanied by reduced pulmonary histopathology in hMPV-infected cotton rats compared to normal animals; however, a delayed increase in pathology and pulmonary chemokine expression was seen in immunosuppressed cotton rats. Prophylactic and therapeutic MPV467 treatments protected both upper and lower respiratory tracts against hMPV infection. The lung pathology and pulmonary expression of IP-10 and MIP-1α mRNA were reduced by therapeutic MPV467 administration. These results indicate that immunosuppressed cotton rats represent a useful model for studying hMPV pathogenesis and for evaluating therapeutics that could alleviate hMPV-induced disease in immunocompromised subjects.

SARS-CoV-2 infection augments species- and age-specific predispositions in cotton rats.

Heterogeneity of COVID-19 manifestations in human population is vast, for reasons unknown. Cotton rats are a clinically relevant small animal model of human respiratory viral infections. Here, we demonstrate for the first time that SARS-CoV-2 infection in cotton rats affects multiple organs and systems, targeting species- and age-specific biological processes. Infection of S. fulviventer, which developed a neutralizing antibody response and were more susceptible to SARS-CoV-2 replication in the upper respiratory tract, was accompanied by hyperplasia of lacrimal drainage-associated lymphoid tissue (LDALT), a first known report of mucosa-associated lymphoid tissue activation at the portal of SARS-CoV-2 entry. Although less permissive to viral replication, S. hispidus showed hyperplasia of bone marrow in the facial bones and increased pulmonary thrombosis in aged males. Augmentation of these features by SARS-CoV-2 infection suggests a virus-induced breach in regulatory mechanisms which could be devastating for people of all ages with underlying conditions and in particular for elderly with a multitude of ongoing disorders.

Correlative outcomes of maternal immunization against RSV in cotton rats.

Maternal anti-respiratory syncytial virus (RSV) antibodies protect neonates from RSV disease throughout first weeks of life. Previous studies of maternal immunization in cotton rats showed that a single immunization during pregnancy of RSV-primed dams with virus-like particles (VLPs) assembled with pre-fusion F protein and the wild type G protein boosted their RSV serum antibody concentration and protected pups early in life against RSV challenge. We extended these findings by evaluating responses to RSV infection in litters from two consecutive pregnancies of immunized dams. Using an RSV-primed population of VLP-vaccinated and unvaccinated dams, we defined correlations between dams' and litters' RSV neutralizing antibodies (NA); between litters' NA and protection; and between litter's NA and their lung expression of selected cytokines, of a first or of a second pregnancy. Lung pathology was also evaluated. We found positive correlation between the NA titers in the dams at delivery and the NA in their first and second litters and negative correlations between the litters' NA and protection from RSV challenge. Vaccination of dams modulated the mRNA expression for IFNγ and IL-6 and lung pathology in the first and in the second litter at different times after birth, even in the absence of detectable NA. Maternal RSV vaccination enhanced the levels of antibodies transferred to offspring and their protection from challenge. Importantly, maternal vaccination also impacted the immunological and inflammatory response of the offspring's lungs well into maturity, and after the antiviral effect of maternally transferred NA waned or was no longer detectable.

Evidence for Interplay Between the Renin-Angiotensin System and Toll-Like Receptor 4 Signaling Pathways in the Induction of Virus-Induced Acute Lung Injury.

Dedication: This article is dedicated to Howard Young, an exceptional scientist who has provided outstanding mentorship to many postbaccalaureates, graduate students, and postdoctoral fellows during his career. Howard has been a colleague to many and was never tired of learning new things. He has brought "thinking out of the box" to the level of an art form and has always provided thoughtful and constructive suggestions to those who have sought his counsel. I am personally greatly indebted to Howard for his guidance in molecular biology over the past 30 years, and hope that we will continue to share a passion for learning and mentoring others for years to come. Thank you, Howard! -Stephanie N. Vogel The SARS-CoV-2 pandemic has led to an unprecedented explosion in studies that have sought to identify key mechanisms that underlie the ravaging aspects of this disease on individuals. SARS-CoV-2 virus gains access to cells by (1) binding of the viral spike (S) protein to cell-associated angiotensin-converting enzyme 2 (ACE2), a key receptor in the renin-angiotensin system (RAS), followed by (2) cleavage of S protein by a cellular serine protease ("S protein priming") to facilitate viral entry. Dysregulation of the RAS system has been implicated in the spectrum of clinical symptoms associated with SARS-CoV-2, including hypercytokinemia, elevated markers of endothelial injury and thrombosis, and both localized and systemic inflammation. However, the underlying mechanisms have yet to be fully delineated.

Species-specific transcriptomic changes upon respiratory syncytial virus infection in cotton rats.

The cotton rat (Sigmodon) is the gold standard pre-clinical small animal model for respiratory viral pathogens, especially for respiratory syncytial virus (RSV). However, without a reference genome or a published transcriptome, studies requiring gene expression analysis in cotton rats are severely limited. The aims of this study were to generate a comprehensive transcriptome from multiple tissues of two species of cotton rats that are commonly used as animal models (Sigmodon fulviventer and Sigmodon hispidus), and to compare and contrast gene expression changes and immune responses to RSV infection between the two species. Transcriptomes were assembled from lung, spleen, kidney, heart, and intestines for each species with a contig N50 > 1600. Annotation of contigs generated nearly 120,000 gene annotations for each species. The transcriptomes of S. fulviventer and S. hispidus were then used to assess immune response to RSV infection. We identified 238 unique genes that are significantly differentially expressed, including several genes implicated in RSV infection (e.g., Mx2, I27L2, LY6E, Viperin, Keratin 6A, ISG15, CXCL10, CXCL11, IRF9) as well as novel genes that have not previously described in RSV research (LG3BP, SYWC, ABEC1, IIGP1, CREB1). This study presents two comprehensive transcriptome references as resources for future gene expression analysis studies in the cotton rat model, as well as provides gene sequences for mechanistic characterization of molecular pathways. Overall, our results provide generalizable insights into the effect of host genetics on host-virus interactions, as well as identify new host therapeutic targets for RSV treatment and prevention.

Protection against influenza-induced Acute Lung Injury (ALI) by enhanced induction of M2a macrophages: possible role of PPARγ/RXR ligands in IL-4-induced M2a macrophage differentiation.

Many respiratory viruses cause lung damage that may evolve into acute lung injury (ALI), a cytokine storm, acute respiratory distress syndrome, and ultimately, death. Peroxisome proliferator activated receptor gamma (PPARγ), a member of the nuclear hormone receptor (NHR) family of transcription factors, regulates transcription by forming heterodimers with another NHR family member, Retinoid X Receptor (RXR). Each component of the heterodimer binds specific ligands that modify transcriptional capacity of the entire heterodimer by recruiting different co-activators/co-repressors. However, the role of PPARγ/RXR ligands in the context of influenza infection is not well understood. PPARγ is associated with macrophage differentiation to an anti-inflammatory M2 state. We show that mice lacking the IL-4Rα receptor, required for M2a macrophage differentiation, are more susceptible to mouse-adapted influenza (A/PR/8/34; "PR8")-induced lethality. Mice lacking Ptgs2, that encodes COX-2, a key proinflammatory M1 macrophage mediator, are more resistant. Blocking the receptor for COX-2-induced Prostaglandin E2 (PGE2) was also protective. Treatment with pioglitazone (PGZ), a PPARγ ligand, increased survival from PR8 infection, decreased M1 macrophage gene expression, and increased PPARγ mRNA in lungs. Conversely, conditional knockout mice expressing PPARγ-deficient macrophages were significantly more sensitive to PR8-induced lethality. These findings were extended in cotton rats: PGZ blunted lung inflammation and M1 cytokine gene expression after challenge with non-adapted human influenza. To study mechanisms by which PPARγ/RXR transcription factors induce canonical M2a genes, WT mouse macrophages were treated with IL-4 in the absence or presence of rosiglitazone (RGZ; PPARγ ligand), LG100754 (LG; RXR ligand), or both. IL-4 dose-dependently induced M2a genes Arg1, Mrc1, Chil3, and Retnla. Treatment of macrophages with IL-4 and RGZ and/or LG differentially affected induction of Arg1 and Mrc1 vs. Chil3 and Retnla gene expression. In PPARγ-deficient macrophages, IL-4 alone failed to induce Arg1 and Mrc1 gene expression; however, concurrent treatment with LG or RGZ + LG enhanced IL-4-induced Arg1 and Mrc1 expression, but to a lower level than in WT macrophages, findings confirmed in the murine alveolar macrophage cell line, MH-S. These findings support a model in which PPARγ/RXR heterodimers control IL-4-induced M2a differentiation, and suggest that PPARγ/RXR agonists should be considered as important tools for clinical intervention against influenza-induced ALI.

Human Adenovirus Type 4 Comprises Two Major Phylogroups with Distinct Replicative Fitness and Virulence Phenotypes.

Human adenovirus type 4 (HAdV-E4) is the only type (and serotype) classified at present within species Human mastadenovirus E that has been isolated from a human host. Recent phylogenetic analysis of whole-genome sequences of strains representing the spectrum of intratypic genetic diversity described to date identified two major evolutionary lineages designated phylogroups (PGs) I and II and validated the early clustering of HAdV-E4 genomic variants into two major groups by low-resolution restriction fragment length polymorphism analysis. In this study, we expanded our original analysis of intra- and inter-PG genetic variability and used a panel of viruses representative of the spectrum of genetic diversity described for HAdV-E4 to examine the magnitude of inter- and intra-PG phenotypic diversity using an array of cell-based assays and a cotton rat model of HAdV respiratory infection. Our proteotyping of HAdV-E strains using concatenated protein sequences in selected coding regions including E1A, E1B-19K and -55K, DNA polymerase, L4-100K, various E3 proteins, and E4-34K confirmed that the two clades encode distinct variants/proteotypes at most of these loci. Our in vitro and in vivo studies demonstrated that PG I and PG II differ in their growth, spread, and cell-killing phenotypes in cell culture and in their pulmonary pathogenic phenotypes. Surprisingly, the differences in replicative fitness documented in vitro between PGs did not correlate with the differences in virulence observed in the cotton rat model. This body of work is the first reporting phenotypic correlates of naturally occurring intratypic genetic variability for HAdV-E4. IMPORTANCE Human adenovirus type 4 (HAdV-E4) is a prevalent causative agent of acute respiratory illness of variable severity and of conjunctivitis and comprises two major phylogroups that carry distinct coding variations in proteins involved in viral replication and modulation of host responses to infection. Our data show that phylogroup (PG) I and PG II are intrinsically different regarding their ability to grow and spread in culture and to cause pulmonary disease in cotton rats. This is the first report of phenotypic divergence among naturally occurring known genetic variants of an HAdV type of medical importance. This research reveals readily detectable phenotypic differences between strains representing phylogroups I and II, and it introduces a unique experimental system for the elucidation of the genetic basis of adenovirus fitness and virulence and thus for increasing our understanding of the implications of intratypic genetic diversity in the presentation and course of HAdV-E4-associated disease.

Evolution of protection after maternal immunization for respiratory syncytial virus in cotton rats.

Maternal anti-respiratory syncytial virus (RSV) antibodies acquired by the fetus through the placenta protect neonates from RSV disease through the first weeks of life. In the cotton rat model of RSV infections, we previously reported that immunization of dams during pregnancy with virus-like particles assembled with mutation stabilized pre-fusion F protein as well as the wild type G protein resulted in robust protection of their offspring from RSV challenge. Here we describe the durability of those protective responses in dams, the durability of protection in offspring, and the transfer of that protection to offspring of two consecutive pregnancies without a second boost immunization. We report that four weeks after birth, offspring of the first pregnancy were significantly protected from RSV replication in both lungs and nasal tissues after RSV challenge, but protection was reduced in pups at 6 weeks after birth. However, the overall protection of offspring of the second pregnancy was considerably reduced, even at four weeks of age. This drop in protection occurred even though the levels of total anti-pre-F IgG and neutralizing antibody titers in dams remained at similar, high levels before and after the second pregnancy. The results are consistent with an evolution of antibody properties in dams to populations less efficiently transferred to offspring or the less efficient transfer of antibodies in elderly dams.

Targeting TLR4 Signaling to Blunt Viral-Mediated Acute Lung Injury.

Respiratory viral infections have been a long-standing global burden ranging from seasonal recurrences to the unexpected pandemics. The yearly hospitalizations from seasonal viruses such as influenza can fluctuate greatly depending on the circulating strain(s) and the congruency with the predicted strains used for the yearly vaccine formulation, which often are not predicted accurately. While antiviral agents are available against influenza, efficacy is limited due to a temporal disconnect between the time of infection and symptom development and viral resistance. Uncontrolled, influenza infections can lead to a severe inflammatory response initiated by pathogen-associated molecular patterns (PAMPs) or host-derived danger-associated molecular patterns (DAMPs) that ultimately signal through pattern recognition receptors (PRRs). Overall, these pathogen-host interactions result in a local cytokine storm leading to acute lung injury (ALI) or the more severe acute respiratory distress syndrome (ARDS) with concomitant systemic involvement and more severe, life threatening consequences. In addition to traditional antiviral treatments, blocking the host's innate immune response may provide a more viable approach to combat these infectious pathogens. The SARS-CoV-2 pandemic illustrates a critical need for novel treatments to counteract the ALI and ARDS that has caused the deaths of millions worldwide. This review will examine how antagonizing TLR4 signaling has been effective experimentally in ameliorating ALI and lethal infection in challenge models triggered not only by influenza, but also by other ALI-inducing viruses.

Microbial community structure and composition is associated with host species and sex in Sigmodon cotton rats.

BACKGROUND: The cotton rat (genus Sigmodon) is an essential small animal model for the study of human infectious disease and viral therapeutic development. However, the impact of the host microbiome on infection outcomes has not been explored in this model, partly due to the lack of a comprehensive characterization of microbial communities across different cotton rat species. Understanding the dynamics of their microbiome could significantly help to better understand its role when modeling viral infections in this animal model. RESULTS: We examined the bacterial communities of the gut and three external sites (skin, ear, and nose) of two inbred species of cotton rats commonly used in research (S. hispidus and S. fulviventer) by using 16S rRNA gene sequencing, constituting the first comprehensive characterization of the cotton rat microbiome. We showed that S. fulviventer maintained higher alpha diversity and richness than S. hispidus at external sites (skin, ear, nose), but there were no differentially abundant genera. However, S. fulviventer and S. hispidus had distinct fecal microbiomes composed of several significantly differentially abundant genera. Whole metagenomic shotgun sequencing of fecal samples identified species-level differences between S. hispidus and S. fulviventer, as well as different metabolic pathway functions as a result of differential host microbiome contributions. Furthermore, the microbiome composition of the external sites showed significant sex-based differences while fecal communities were not largely different. CONCLUSIONS: Our study shows that host genetic background potentially exerts homeostatic pressures, resulting in distinct microbiomes for two different inbred cotton rat species. Because of the numerous studies that have uncovered strong relationships between host microbiome, viral infection outcomes, and immune responses, our findings represent a strong contribution for understanding the impact of different microbial communities on viral pathogenesis. Furthermore, we provide novel cotton rat microbiome data as a springboard to uncover the full therapeutic potential of the microbiome against viral infections.

Effect of aging on immunogenicity and efficacy of inactivated influenza vaccines in cotton rats Sigmodon hispidus.

Inactivated influenza vaccines are known to be less immunogenic in human elderly in regards to serologic antibody response induced by vaccination. Accumulating evidence, however, points to a comparable effectiveness of influenza vaccines in the young and the elderly individuals. In the current study, we assessed immunogenicity and effectiveness of trivalent inactivated vaccine FluLaval in young and aged cotton rats Sigmodon hispidus and found that while serologic response to immunization was indeed reduced in older animals, comparable protection against influenza infection was afforded by prime-boost vaccination in both young and aged cotton rats. Both hemagglutination inhibition (HAI) titers and seroconversion rates were lower in the aged animals compared to the young ones. Reduction of viral load in the lung and nose, however, was comparable between young and aged animals vaccinated twice. One-time immunization with FluLaval was less efficacious at protecting the nose of aged animals, indicating that boosting of preexisting immunity can be particularly important for nasal protection in the elderly. Coincidentally, a one-time immunization with FluLaval had a detrimental effect on pulmonary pathology in the young animals, suggesting that boosting of immunity is essential for the young as well. Overall, these results suggest that reduced antibody response to and sufficient efficacy of influenza vaccines in the elderly are not two irreconcilable phenomena and that incomplete immunity to influenza can be detrimental at any age.

A mouse model of human TLR4 D299G/T399I SNPs reveals mechanisms of altered LPS and pathogen responses.

Two cosegregating single-nucleotide polymorphisms (SNPs) in human TLR4, an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I), have been associated with LPS hyporesponsiveness and differential susceptibility to many infectious or inflammatory diseases. However, many studies failed to confirm these associations, and transfection experiments resulted in conflicting conclusions about the impact of these SNPs on TLR4 signaling. Using advanced protein modeling from crystallographic data of human and murine TLR4, we identified homologous substitutions of these SNPs in murine Tlr4, engineered a knock-in strain expressing the D298G and N397I TLR4 SNPs homozygously, and characterized in vivo and in vitro responses to TLR4 ligands and infections in which TLR4 is implicated. Our data provide new insights into cellular and molecular mechanisms by which these SNPs decrease the TLR4 signaling efficiency and offer an experimental approach to confirm or refute human data possibly confounded by variables unrelated to the direct effects of the SNPs on TLR4 functionality.

Comparisons of Antibody Populations in Different Pre-Fusion F VLP-Immunized Cotton Rat Dams and Their Offspring.

Respiratory syncytial virus (RSV) infection poses a significant risk for infants. Since the direct vaccination of infants is problematic, maternal vaccination may provide a safer, more effective approach to their protection. In the cotton rat (CR) model, we have compared the immunization of pregnant CR dams with virus-like particles assembled with the prototype mutation stabilized pre-fusion F protein, DS-Cav1, as well two alternative mutation stabilized pre-fusion proteins (UC-2 F, UC-3 F) and showed that the alternative pre-fusion F VLPs protected the offspring of immunized dams significantly better than DS-Cav1 F VLPs (Blanco, et al. J. Virol. 93: e00914). Here, we have addressed the reasons for this increased protection by characterizing the specificities of antibodies in the sera of both immunized dams and their offspring. The approach was to measure the levels of total anti-pre-F IgG serum antibodies that would block the binding of representative pre-fusion specific monoclonal antibodies to soluble pre-fusion F protein targets. Strikingly, we found that the sera in most offspring of DS-Cav1 F VLP-immunized dams had no mAb D25-blocking antibodies, although their dams had robust levels. In contrast, all offspring of UC-3 F VLP-immunized dams had robust levels of these D25-blocking antibodies. Both sets of pup sera had significant levels of mAb AM14-blocking antibodies, indicating that all pups received maternal antibodies. A lack of mAb D25-blocking antibodies in the offspring of DS-Cav1 F VLP-immunized dams may account for the lower protection of their pups from challenge compared to the offspring of UC-3 F VLP-immunized dams.

Herpes Simplex Virus 1 Induces Brain Inflammation and Multifocal Demyelination in the Cotton Rat Sigmodon hispidus.

Demyelinating central nervous system (CNS) disorders like multiple sclerosis (MS) and acute disseminated encephalomyelitis (ADEM) have been difficult to study and treat due to the lack of understanding of their etiology. Numerous cases point to the link between herpes simplex virus (HSV) infection and multifocal CNS demyelination in humans; however, convincing evidence from animal models has been missing. In this work, we found that HSV-1 infection of the cotton rat Sigmodon hispidus via a common route (lip abrasion) can cause multifocal CNS demyelination and inflammation. Remyelination occurred shortly after demyelination in HSV-1-infected cotton rats but could be incomplete, resulting in "scars," further supporting an association between HSV-1 infection and multifocal demyelinating disorders. Virus was detected sequentially in the lip, trigeminal ganglia, and brain of infected animals. Brain pathology developed primarily on the ipsilateral side of the brain stem, in the cerebellum, and contralateral side of the forebrain/midbrain, suggesting that the changes may ascend along the trigeminal lemniscus pathway. Neurologic defects occasionally detected in infected animals (e.g., defective whisker touch and blink responses and compromised balance) could be representative of the brain stem/cerebellum dysfunction. Immunization of cotton rats with a split HSV-1 vaccine protected animals against viral replication and brain pathology, suggesting that vaccination against HSV-1 may protect against demyelinating disorders.IMPORTANCE Our work demonstrates for the first time a direct association between infection with herpes simplex virus 1, a ubiquitous human pathogen generally associated with facial cold sores, and multifocal brain demyelination in an otherwise normal host, the cotton rat Sigmodon hispidus For a long time, demyelinating diseases were considered to be autoimmune in nature and were studied by indirect methods, such as immunizing animals with myelin components or feeding them toxic substances that induce demyelination. Treatment against demyelinating diseases has been elusive, partially because of their unknown etiology. This work provides the first experimental evidence for the role of HSV-1 as the etiologic agent of multifocal brain demyelination in a normal host and suggests that vaccination against HSV-1 can help to combat demyelinating disorders.

Alternative Virus-Like Particle-Associated Prefusion F Proteins as Maternal Vaccines for Respiratory Syncytial Virus.

Maternal vaccination may be the most effective and safest approach to the protection of infants from respiratory syncytial virus (RSV) infection, a severe acute lower respiratory tract disease in infants and young children worldwide. We previously compared five different virus-like particle (VLP)-associated, mutation-stabilized prefusion F (pre-F) proteins, including the prototype DS-Cav1 F VLPs. We showed that alternative versions of prefusion F proteins have different conformations and induce different populations of anti-F protein antibodies. Two of these alternative pre-F VLPs, the UC-2 F and UC-3 F VLPs, stimulated in mice higher titers of neutralizing antibodies than DS-Cav1 F VLPs (M. L. Cullen, R. M. Schmidt, M. G. Torres, A. A. Capoferri, et al., Vaccines 7:21-41, 2019, https://doi.org/10.3390/vaccines7010021). Here we describe a comparison of these two pre-F VLPs with DS-Cav1 F VLPs as maternal vaccines in cotton rats and report that UC-3 F VLPs significantly increased the neutralizing antibody (NAb) titers in pregnant dams compared to DS-Cav1 F VLPs. The neutralizing antibody titers in the sera of the offspring of the dams immunized with UC-3 F VLPs were significantly higher than those in the sera of the offspring of dams immunized with DS-Cav1 VLPs. This increase in serum NAb titers translated to a 6- to 40-fold lower virus titer in the lungs of the RSV-challenged offspring of dams immunized with UC-3 F VLPs than in the lungs of the RSV-challenged offspring of dams immunized with DS-Cav1 F VLPs. Importantly, the offspring of UC-3 F VLP-immunized dams showed significant protection from lung pathology and from induction of inflammatory lung cytokine mRNA expression after RSV challenge. Immunization with UC-3 F VLPs also induced durable levels of high-titer neutralizing antibodies in dams.IMPORTANCE Respiratory syncytial virus (RSV) is a significant human pathogen severely impacting neonates and young children, but no vaccine exists to protect this vulnerable population. Furthermore, direct vaccination of neonates is likely ineffective due to the immaturity of their immune system, and neonate immunization is potentially unsafe. Maternal vaccination may be the best and safest approach to the protection of neonates through the passive transfer of maternal neutralizing antibodies in utero to the fetus after maternal immunization. Here we report that immunization of pregnant cotton rats, a surrogate model for human maternal immunization, with novel RSV virus-like particle (VLP) vaccine candidates containing stabilized prefusion RSV F proteins provides significant levels of protection of the offspring of immunized dams from RSV challenge. We also found that antibodies induced by VLPs containing different versions of the prefusion F protein varied by 40-fold in the extent of protection provided to the offspring of vaccinated dams upon RSV challenge.

Influenza "Trains" the Host for Enhanced Susceptibility to Secondary Bacterial Infection.

We previously reported that the Toll-like receptor 4 (TLR4) antagonist Eritoran blocks acute lung injury (ALI) therapeutically in mouse and cotton rat models of influenza. However, secondary (2°) bacterial infection following influenza virus infection is associated with excess morbidity and mortality. Wild-type (WT) mice infected with mouse-adapted influenza A/Puerto Rico/8/34 virus (PR8) and, 7 days later, with Streptococcus pneumoniae serotype 3 (Sp3) exhibited significantly enhanced lung pathology and lethality that was reversed by Eritoran therapy after PR8 infection but before Sp3 infection. Cotton rats infected with nonadapted pH1N1 influenza virus and then superinfected with methicillin-resistant Staphylococcus aureus also exhibited increased lung pathology and serum high-mobility-group box 1 (HMGB1) levels, both of which were blunted by Eritoran therapy. In mice, PR8 infection suppressed Sp3-induced CXCL1 and CXCL2 mRNA, reducing neutrophil infiltration and increasing the bacterial burden, all of which were reversed by Eritoran treatment. While beta interferon (IFN-ß)-deficient (IFN-ß-/-) mice are highly susceptible to PR8, they exhibited delayed death upon Sp3 superinfection, indicating that while IFN-ß was protective against influenza, it negatively impacted the host response to Sp3 IFN-ß-treated WT macrophages selectively suppressed Sp3-induced CXCL1/CXCL2 transcriptionally, as evidenced by reduced recruitment of RNA polymerase II to the CXCL1 promoter. Thus, influenza establishes a "trained" state of immunosuppression toward 2° bacterial infection, in part through the potent induction of IFN-ß and its downstream transcriptional regulation of chemokines, an effect reversed by Eritoran.IMPORTANCE Enhanced susceptibility to 2° bacterial infections following infection with influenza virus is a global health concern that accounts for many hospitalizations and deaths, particularly during pandemics. The complexity of the impaired host immune response during 2° bacterial infection has been widely studied. Both type I IFN and neutrophil dysfunction through decreased chemokine production have been implicated as mechanisms underlying enhanced susceptibility to 2° bacterial infections. Our findings support the conclusion that selective suppression of CXCL1/CXCL2 represents an IFN-ß-mediated "training" of the macrophage transcriptional response to TLR2 agonists and that blocking of TLR4 therapeutically with Eritoran after influenza virus infection reverses this suppression by blunting influenza-induced IFN-ß.