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Innately Immune

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by Gertrud U. Rey

It is still not entirely clear why children are less susceptible to severe COVID-19. Early hypotheses included the possibility that children may have a stronger innate immune response, which is the response that occurs upon an initial encounter with a pathogen. Results from a recent study support this hypothesis.

To clarify why children have an enhanced ability to control a SARS-CoV-2 infection, the authors of the study collected nasal samples from SARS-CoV-2-negative and SARS-CoV-2-positive children and adults ranging in age from 4 weeks to 77 years. The presence of viral RNA in samples from SARS-CoV-2-positive participants was confirmed by PCR. The samples were also analyzed for the presence of different cell types using single cell RNA sequencing, a method that reveals the RNA expression profiles of individual cells. The authors detected 33 different cell types in the upper respiratory tract of all tested individuals, including 21 immune and 12 epithelial cell subtypes. The differences in the cell compositions of children and adults were quite dramatic – while nasal samples from healthy adults rarely contained immune cells, samples from children contained high levels of almost every immune cell subset, with neutrophils representing a substantial portion of the cell population. Neutrophils are an essential part of the innate immune system because they accumulate quickly at a site of infection, where they ingest pathogens and recruit and activate other immune cells.

Despite this difference in cell composition in the nasal mucosa of children and adults, the expression level of ACE2 (the SARS-CoV-2 binding target), was similar in both age groups. This result is contrary to previous suggestions that children may express less ACE2, but it is consistent with reports indicating that the frequency of SARS-CoV-2 infection in children is similar to that of adults.

Sentinel cells of the innate immune system recognize invading pathogens by sensing structurally conserved molecular motifs in infectious microbes. This sensing occurs through various pattern recognition receptors on or in the immune cells present in most tissues, like the well-characterized RIG-I-like receptors. Together with two other proteins, MDA5 and LGP2, RIG-I-like receptors detect the presence of viral RNA inside our cells and trigger a cascade of events that mobilize immune cells such as macrophages, neutrophils, and dendritic cells to the site of infection. Once there, these immune cells produce pro-inflammatory signaling proteins known as cytokines, which then cue other responses and prime adaptive T and B cells for future functions. Sensing of viral RNA by RIG-I and MDA5 initiates the production of a cytokine called interferon, a signaling protein that triggers downstream protective defenses.

When the authors compared the baseline expression of RIG-I-, MDA5-, and LGP2-encoding genes in the upper respiratory tract epithelial cells of healthy children and adults, they found that healthy children expressed significantly higher levels of these genes compared to healthy adults and SARS-CoV-2-positive adults who were in the early phase of infection. Samples from healthy children also contained a subpopulation of cytotoxic T cells that was absent in adults, and these T cells produced high levels of interferon gamma, a cytokine that inhibits viral replication and induces macrophages to engulf and digest pathogens.

When children became infected with SARS-CoV-2, they produced significantly higher levels of interferon gamma compared to SARS-CoV-2-positive adults, both in the early and later phases of infection. This observation is particularly interesting when considering that impaired interferon responses are a hallmark of severe COVID-19 and that SARS-CoV-2 is highly susceptible to interferon treatment. Furthermore, samples from SARS-CoV-2-infected children contained a subpopulation of SARS-CoV-2-specific memory T cells that was nearly absent in adults, suggesting that children might have an increased ability to respond to future SARS-CoV-2 reinfections. 

The increased numbers of innate immune cells and increased expression of pattern recognition receptor genes in the upper airways of children may facilitate a more efficient innate response to SARS-CoV-2 infection, leading to reduced viral replication and faster clearance of virus. This type of innate immune response seems to be delayed in older adults, and in an effort to “catch up,” may result in excessive inflammation, thereby ultimately causing more severe damage. Although there are likely more factors at play, this study brings us one step closer to understanding why COVID-19 is generally less severe in children.

Defective genomes modulate respiratory syncytial virus pathogenesis

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During viral replication, defective genomes may arise that lack essential sequences. These so-called defective genomes cannot replicate unless they are in the same cell as a helper virus. Defective genomes play a role in modulating pathogenesis of respiratory syncytial virus in humans.

Copy-back defective viral genomes (cbDVGs) of RSV arise when the viral RNA polymerase halts and then turns around and begins copying the nascent strand (illustrated below – image credit). Because the cbDVGs have strands of both (+) and (-) polarity it is possible to design primers to amplify them by RT-PCR.

It has been suspected that defective viral genomes might modulate viral pathogenesis because they are efficient inducers of interferon. In a study of 122 hospitalized pediatric patients with RSV infection, the presence of cbDVGs was associated with higher viral loads and longer hospitalizations. Furthermore, patients harboring cbDVGs had higher expression of pro-inflammatory cytokine genes.

Unexpectedly, non-hospitalized patients with confirmed RSV infection also had high levels of cbDVGs and high viral loads. Analysis of the kinetics of cbDVG production revealed that when cbDVGs arise early in infection, less severe symptoms present. In contrast, when cbDVGs arise later in infection, higher viral loads and more severe disease occur. The latter patients also produce higher levels of pro-inflammatory cytokines which likely cause immunopathogenesis.

An interpretation of these findings is that when cbDVGs arise early in infection, they induce antiviral immune responses that limit viral replication and disease. When cbDVGs arise later, the virus has already reproduced to high levels, leading to more severe disease. The factors that regulate cbDVGs production are not known, but could involve sex, age, immune status, and the levels of the defective genomes in the virus inoculum.

These results are of interest because it has not been possible to predict which RSV infected patients will develop severe disease. It might be possible to use levels of cbDVGs to forecast clinical outcome.

When defective viral particles were discovered many years ago, Alice Huang proposed that they might modulate viral pathogenesis. It was not until the 1990s that the technology became available to test this hypothesis. The results with RSV suggest that whether cbDVGs are beneficial or detrimental depends on when they arise in infection.

A herpesvirus associated with female infertility

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HHV-6Viruses that replicate in the male or female reproductive tract are considered to be potential causes of human infertility. Several herpesviruses have been implicated in male infertility, and now human herpesvirus 6A (HHV-6A) has been found in endometrial cells of women with unexplained infertility (paper link).

HHV-6 was only recently discovered (1986) and is now known to occur as two variants, HHV-6A and HHV-6B. The latter is a major cause of exanthem subitum, a rash of infants, but no disease has been clearly associated with HHV-6A. These viruses are transmitted to infants early in life via saliva, from mother to child, from siblings, or from other infants at day care centers. Seroprevalence studies indicate that almost all children are infected with these viruses by 2 years of age.

To determine if HHV-6 might be a cause of infertility, a study (paper link) was conducted of 30 women with unexplained primary fertility, and 36 women with at least one previous pregnancy. HHV-6B DNA was detected in PBMC from both infertile and fertile groups (25 and 28%, respectively); HHV-6A DNA was not detected. In contrast, endometrial epithelial cells from 13/30 (43%) infertile women were positive for HHV-6A DNA; this viral DNA was not detected in endometrium of fertile women. When placed in culture, endometrial epithelial cells produced viral early and late proteins, suggesting the presence of infectious virus.

Presence of HHV-6A DNA in endometrial epithelial cells was also associated with an altered hormonal and immune environment. Estradiol levels were higher in infected versus uninfected infertile women. The authors suggest that higher levels of this hormone could be involved in allowing HHV-6A infection of the endometrium.

Levels of a specific type of uterine NK cell were lower in HHV-6A positive women, and IL-10 (a Th2 cytokine) was elevated while IFN-gamma (a Th1 cytokine) was decreased. There were no differences in the levels of these cells and cytokines in peripheral blood. These changes are consistent with an increase in the ratio of Th1/Th2 responses that has been documented in female infertility.

The authors also observed enhanced endometrial NK cell responses to HHV-6A in infected but not uninfected women, together with an increase in the number of these cells that are activated when cultured with HHV-6A infected cells.

I wonder what was the source of HHV-6A in the endometrium, as the virus was not detected in blood. Was the infection recently acquired, or did it occur years before, with the virus establishing a chronic infection in the uterus?

The results suggest that HHV-6A infection of the endometrium triggers an abnormal NK cell and cytokine profile, which in turn leads to a uterine environment that is not compatible with fertility. The results need to be confirmed with studies of additional fertile and infertile women. It would also be useful to have an animal model of HHV-6A infection of the endometrium, which could lead to mechanistic work to determine how virus infection causes infertility.

Image: Electron micrograph of HHV-6 (image credit)

TWiV 214: This is your brain on polyomavirus

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On episode #214 of the science show This Week in Virology, Vincent, Alan, and Kathy discuss how coagulation factor X binding to adenovirus activates the innate immune system, and a novel polyomavirus associated with brain tumors in raccoons.

You can find TWiV #214 at www.microbe.tv/twiv.

The inflammatory response

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neutrophil-migrationDuring the earliest stages of a virus infection, cytokines are produced when innate immune defenses are activated. The rapid release of cytokines at the site of infection initiates new responses with far-reaching consequences that include inflammation.

One of the earliest cytokines produced is tumor necrosis factor alpha (TNF-α), which is synthesized by activated monocytes and macrophages. This cytokine changes nearby capillaries so that circulating white blood cells can be easily brought to the site of infection. TNF-α can also bind to receptors on infected cells and induce an antiviral response. Within seconds, a series of signals is initiated that leads to cell death, an attempt to prevent the spread of infection.

Inflammation is a very prominent response to TNF-α. There are four typical signs of inflammation: erythema (redness), heat, swelling, and pain. These are a consequence of increased blood flow and capillary permeability, the influx of phagocytic cells, and tissue damage. Increased blood flow is caused by constriction of the capillaries that carry blood away from the infected area, and leads to engorgement of the capillary network. Erythema and an increase in tissue temperature accompany capillary constriction. In addition, the permeability of capillaries increases, allowing cells and fluid to leave and enter the surrounding tissue. These fluids have a higher protein content than the fluids normally found in tissues, causing swelling.

Another feature of inflammation is the presence of immune cells, largely mononuclear phagocytes, which are attracted to the infected area by cytokines. Neutrophils are one of the earliest types of phagocytic cells that enter a site of infection, and are classic markers of the inflammatory response (illustrated). These cells are abundant in the blood, and usually absent from tissues. Together with infected cells, dendritic cells, and macrophages, they produce cytokines that can further shape the response to infection, and also modulate the adaptive response that may follow.

The precise nature of the inflammatory response depends upon the virus and the tissue that is infected. Viruses that do not kill cells – noncytopathic viruses – do not induce a strong inflammatory response. Because the cells and proteins of the inflammatory response come from the bloodstream, tissues with reduced access to the blood do not undergo the destruction associated with inflammation. However, the outcome of infection in such ‘privileged’ sites – the brain, for example – may be very different compared with other tissues.

As expected, the inflammatory response is highly regulated. One of the critical components is the ‘inflammasome’ – very large cytoplasmic structure with properties of pattern receptors and initiators of signaling (e.g. MDA-5 and RIG-I). Recent experimental findings demonstrate that the inflammasome is critical in innate immune response to influenza virus infection, and in moderating lung pathology in influenza pneumonia.

Thomas, P., Dash, P., Aldridge Jr., J., Ellebedy, A., Reynolds, C., Funk, A., Martin, W., Lamkanfi, M., Webby, R., & Boyd, K. (2009). The Intracellular Sensor NLRP3 Mediates Key Innate and Healing Responses to Influenza A Virus via the Regulation of Caspase-1 Immunity, 30 (4), 566-575 DOI: 10.1016/j.immuni.2009.02.006

Allen, I., Scull, M., Moore, C., Holl, E., McElvania-TeKippe, E., Taxman, D., Guthrie, E., Pickles, R., & Ting, J. (2009). The NLRP3 Inflammasome Mediates In Vivo Innate Immunity to Influenza A Virus through Recognition of Viral RNA Immunity, 30 (4), 556-565 DOI: 10.1016/j.immuni.2009.02.005