Early viral exposure in mice weakens vaccine response, raising questions for human studies

In a preprint* research paper recently uploaded to the bioRxiv server, researchers at Washington University investigated how the exposome can mediate immune function in house mice, one of the most widely used model systems for in vivo immunological experimentation. Mice were sequentially inoculated with six different viral pathogens from early life (neonatal) stages, following which their immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was measured. Their results suggest that viral exposure during early life stages can substantially reduce antibody responses to vaccination, making prior pathogen exposure an essential consideration in murine model vaccine studies.

Study: Sequential early-life viral infections modulate the microbiota and adaptive immune responses to systemic and mucosal vaccination. Image Credit: Christoph Burgstedt / Shutterstock

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

The exposome

Murine models, especially those using house mice (Mus musculus), are some of the most commonly used in vivo systems for immunological and biomedical research. Mice have a short generation time, are easy to rear and experiment upon under controlled laboratory environments, well-characterized phenotypically and physiologically represent human immunological responses. This makes these animals versatile and reliable model systems to test vaccines, pharmaceutical drugs, and immunological modalities.

Recent research has identified a potential oversight in the conventional use of murine models – laboratory mice are reared in tightly controlled, specific pathogen-free (SPF) animal facilities, thereby preventing their exposure to natural microbes that humans and wild mice encounter under normal conditions. Despite genotypic and immune system structure and function commonalities between laboratory mice and their wild counterparts, a growing body of evidence elucidates that pathogen exposure in the former invokes different immunological responses than those observed in the latter.

The exposome is a relatively novel concept, defined by Miller and Jones in 2014 as the “cumulative measure of environmental influences and associated biologic responses throughout the life span, including exogenous exposures and endogenous processes.” The exposome is thus the sum of all external and internal chemical, physical, biological, and social factors influencing health. Since wild mice are exposed to a plethora of naturally occurring microbial influences that their laboratory-raised counterparts never encounter, the exposome concept hypothesizes that these cohorts would depict observable differences in their immune response to viral or pathogenic inoculation.

Scant research into this association has hitherto been unable to experimentally verify this expectation, with studies being unable to establish differential immunological trends between wild- and laboratory-reared animals. These studies have focused on adult mice, which may fail to account for early-life changes that microbial exposure might modulate. There exists a need for studies to test how developmental exposure might ‘prime’ the immune systems of mice, potentially differing from observations from adult exposure where pathogenic priming may not alter immune function.

“In an effort to develop a tractable sequential infection model for broad laboratory use, as well as to further describe the microbial and immune changes that result from sequential microbial exposures, we devised a virus-only sequential infection model beginning in early life that is completed by 6 weeks of age, allowing mice to be used for further experiments in a rapid and well-controlled manner.”

About the study

The present study aims to devise a murine immune system priming model that is initiated in the neonatal stage and persists across life. Sequential viral inoculation is expected to cause observable changes in immune cell population composition and antibody and cytokine expression levels. If successful, this would form the basis for a traceable ‘humanized’ model of immune response that better reflects the real-life outcomes of vaccination.

Researchers used a case-control study design wherein wild-type (WT) C57BL/6J mice were allowed to mate. Their progeny were divided into the sequentially infected case-cohort and controls raised under conventional SPF methodology. Case mice were inoculated at age seven days using six viral pathogens – murine rotavirus strain (MRV), murine gamma-herpesvirus 68 (MHV68), murine norovirus strain CR6 (MNV), influenza virus strain PR8 (IAV), coxsackievirus B3 (CVB3), and murine astrovirus (MAstV). Inoculation was conducted sequentially at one-week intervals.

Fecal and blood samples were periodically collected for immunological measurements. Following final inoculum exposure, mice were allowed four weeks of recovery, after which ChAd-SARS-CoV-2-S viral particles were introduced via intramuscular injection to both cohorts to simulate pathogenic vaccination.

To investigate complete blood counts (CBC) and differential white blood cell (WBC) composition between test and control mice cohorts, hematological analysis was used. Enzyme-linked immunoassay (ELISA) experiments were used to identify antibody specificity following vaccination. Flow cytometry was employed to identify and characterize splenocytes, tissue cells, and peripheral blood leukocytes. Multiplex immunoassays and mouse Antibody Isotyping Panel (AIP) were used to measure serum chemokines, antibodies, and cytokines. Intercellular cytokine staining and peptide restimulation assay were conducted to verify these outcomes.

16S rRNA gene Illumina sequencing was carried out to identify and quantify viral DNA present in mouse fecal samples. Finally, statistical significance testing was employed to characterize and discuss differences in observed outcomes between case and control mice.

Study findings

“We sought to develop an “immunologically-mature” murine model in a genetically defined background by utilizing a well-controlled series of microbial exposures, focused exclusively on viruses, that could be rapidly administered to permit further experimental intervention by the age of 10 weeks.”

Sequential viral exposure was found to create a persistent pro-inflammatory host environment in case mice compared to their control counterparts. Global immunological changes were observed in case mice with hematological analysis, revealing that at week 10, leukocytes were significantly unregulated when compared to controls raised under aseptic conditions. While WBC proportions remained unchanged, their absolute numbers increased substantially in case mice. Serum cytokine analysis revealed drastic increases in pro-inflammatory cytokines interleukin (IL)-6, interferon (IFN)-g, and tumor necrosis factor at nine weeks of exposure, which together demonstrate enhanced immune response in viral-exposed mice.

Sequential viral infection was found to control the circulating and tissue-resident components of adaptive immunity in case mice. Viral exposure was additionally observed to modulate intestinal microbiome composition. In contrast, SPF mice were found to show minimal variation in gut microbiome composition. Inoculation with the SARS-CoV-2 vaccine was found to limit antibody response and unregulate T-cell expression in the case-cohort, reducing symptomatic presentation and potentially reducing vaccine efficacy.

Conclusions

This preprint presents the first evidence of the effects of viral exposure across life stages, especially in neonatal and early life periods. Researchers sequentially exposed one-week-old mice to six viral pathogens, following which intramuscular administration of a SARS-CoV-2 vaccine was undertaken. Unlike previous work, substantial differences between cytokines, WBCs, and other immune-modulating components were observed between sequentially inoculated mice and their SPF counterparts.

“Rodent models with heightened microbial exposures, either via cohousing with “dirty” mice or via sequential infection, have been shown to exhibit diminished humoral responses to vaccination.”

Study findings mirror these results and suggest that while innate and adaptive immunity is stronger in mice exposed to viral exposure in early life, their response to vaccination is blunted, highlighting that in vivo vaccine testing in SPF murine models overestimates vaccine efficacy, a trend that likely extends to humans.

“Overall, the results of this study indicate that sequential viral infections modulate the microbiota and lead to changes in the immune system that dampen specific adaptive responses to systemic and mucosal vaccination. This study highlights the importance of early-life microbial exposure and its impact on the immune system and gut microbiota. Sequential infection provides a powerful model for a matured immune system that can be readily leveraged for immunology, virology, and vaccine studies.”

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.