virology blog

About viruses and viral disease

Gertrud Rey

Dynamics of SARS-CoV-2 Vaccine-Induced Antibody Immunity

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Gertrud U. Rey

Vaccination against the vaccine-preventable diseases is preferable to natural infection because it prevents illness and the long-term effects associated with many infections; and in most cases, it also leads to better immunity. In the case of immunity induced by SARS-CoV-2 vaccination, it is slowly becoming clear that the immunity is of overall better quality than that induced by COVID-19.

Vaccination leads to production of polyclonal antibodies, which are derived from many different types of B cells and may target many different portions of an antigen (i.e., “epitopes”). Isolation of B cells from a blood sample allows one to artificially produce monoclonal antibodies, which are derived from a single type of B cell and target a single epitope. Using blood samples from 6 SARS-CoV-2 mRNA vaccine recipients and 30 COVID-19 survivors, the authors of a recent publication compared the dynamics of vaccine-induced antibody immunity to those resulting from natural infection. A comparison of total antibodies between the two groups of people revealed that polyclonal antibody levels in vaccinees were generally higher than those in COVID-19 survivors. However, when the authors tested the antibodies from vaccinees for SARS-CoV-2 neutralizing activity, only a minority were neutralizing, including antibodies that target the receptor-binding domain (RBD) on the SARS-CoV-2 spike protein.

To see whether any of the neutralizing antibodies from vaccinees were capable of binding to the RBD, the authors performed competition experiments, where they exposed the antibodies to the SARS-CoV-2 RBD alone, or to RBD pre-mixed with its binding target – the human ACE2 receptor. This experiment revealed that increasing concentrations of ACE2 led to decreased antibody binding, suggesting that the antibodies compete with the ACE2 receptor for binding to the RBD, and that they neutralize the virus by inhibiting its binding to ACE2.

A plasmablast is a type of B cell that differentiates from an immature B cell into a mature plasma cell, which can produce large amounts of a specific antibody. Although they are short-lived, plasmablasts also make antibodies, and are typically abundant and easy to isolate from peripheral blood. Analysis of monoclonal antibodies produced from the plasmablasts of vaccinated individuals showed that a substantial number of these antibodies also bound the N-terminal domain of the SARS-CoV-2 spike protein in addition to the RBD, suggesting that these two epitopes co-dominate as antibody targets.

“Original antigenic sin” is a phenomenon initially discovered with influenza viruses, where the immune system mounts the strongest response to the first version of one particular antigen encountered (i.e., the first virus variant with which one is infected in life). Similar observations have been made in the context of COVID-19, with natural SARS-CoV-2 infection purportedly also substantially boosting antibodies against seasonal β-coronaviruses OC43 and HKU1. To see if SARS-CoV-2 vaccination produces a similar effect, the authors analyzed the specificity of antibodies isolated from vaccine recipients before and after vaccination against the spike protein of α-coronaviruses 229E and NL63 and β-coronaviruses OC43 and HKU1. Although vaccination did not increase titers of pre-existing antibodies against 229E and NL63 viruses, it did increase titers against OC43 and HKU1, consistent with what happens during natural SARS-CoV-2 infection. These results suggest that some of the vaccine-induced antibody responses may be biased by pre-existing immunity to other circulating human β-coronaviruses.

The authors also determined whether plasmablast-derived monoclonal antibodies and polyclonal antibodies from the sera of COVID-19 survivors and vaccinated individuals could bind to the RBDs of different SARS-CoV-2 variants. Although complete loss of binding was rare, antibodies from COVID-19 survivors varied widely in their ability to bind to different variants. Antibodies isolated from vaccinees varied less, depending on the variant. However, for most plasmablast-derived monoclonal antibodies from vaccinees, there was no impact on binding to RBDs in the binding assay used, regardless of the variant.

This study raises several interesting points. First, the ability of SARS-CoV-2 vaccination to boost pre-existing antibodies against HKU1 and OC43 supports the premise for a universal β-coronavirus vaccine, several of which are already in development. Second, the ability of the RBD and N-terminal domain to co-dominate in eliciting spike-specific antibodies justifies the use of vaccines containing the entire spike protein, rather than just the RBD. Third, although it is surprising that vaccination induced so few neutralizing antibodies, the role of non-neutralizing antibodies in protection against disease and infection is currently unknown. Considering that vaccination is obviously so protective against disease and asymptomatic infection, the authors speculate that the antibodies induced by vaccination could have protective functions outside of neutralization. Lastly, the fact that vaccination-induced antibodies fluctuate little, if at all, in their ability to bind the RBDs of different variants compared to antibodies induced by natural infection, suggests that vaccination is more likely to protect from different variants than natural infection. Collectively, these findings further highlight the importance of vaccination for ending this pandemic.

When Viruses Collide with Parasitic Worms

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by Gertrud U. Rey

The absence of infection with some parasitic worms (also known as helminths) often coincides with the development of asthma, allergy, and autoimmune disorders, suggesting that these worms may have co-evolved a commensal relationship with their human hosts. However, infection with these worms may also lead to an impaired immune response against pathogenic infections by other parasites, bacteria, and some viruses. 

Based on such observations, the authors of a recent study explored the effect of infection with Heligmosomoides polygyrus bakeri (Hpb) – a naturally occurring intestinal roundworm of rodents – on West Nile virus (WNV) infection outcome in mice. WNV is a member of the Flavivirus genus, along with Dengue virus, Zika virus, yellow fever virus, tick-borne encephalitis virus, and Powassan virus. These viruses all infect their human hosts through the bite of an arthropod and ultimately target cells of the central nervous system.

To assess the effect of intestinal Hpb infection on the pathogenesis of WNV, the authors used three groups of mice: the first group was only fed Hpb larvae to initiate an Hpb infection, the second group was only infected with WNV by inoculation in the foot pad, and the third group was infected with both Hpb and WNV. Infection with Hpb alone had no adverse effect on body weight or survival of the mice, and infection with WNV alone caused moderate weight loss and 86% survival. In contrast, infection with both Hpb and WNV led to greater weight loss and only 28% survival. To determine the reason for this enhanced pathogenicity, the mice were euthanized, and different organs were assessed for viral titers. Mice infected only with WNV, or with both WNV and Hpb, had similar levels of virus in the spleen, an organ that filters the blood and produces immune cells in response to the presence of infectious pathogens. However, mice infected with both Hpb and WNV had higher levels of virus in different parts of the colon and the mesenteric lymph nodes, which serve as a firewall to prevent systemic spread of microorganisms. The presence of such high viral titers in these regions suggested that co-infection with Hpb and WNV leads to the infection of cells in gut-proximal places.

To determine which gut cells are targeted by WNV during a Hpb/WNV co-infection, the authors stained pieces of gut tissue from Hpb/WNV co-infected mice with a combination of fluorescent antibodies that bind WNV antigens or neuronal cell markers. Analysis of these tissues by fluorescence microscopy revealed that co-infected animals have higher levels of viral antigens in intestinal neurons compared to control animals infected only with Hpb or WNV, suggesting that infection with both Hpb and WNV increases the frequency of infection of gut neuronal cells. Microscopic analysis also revealed certain gut abnormalities in co-infected animals, including changes in the composition of intestinal villi, which are small, finger-like projections that extend into the lumen of the small intestine. Co-infected animals had shortened villi, a phenomenon typically associated with pathology and inflammation. Co-infected animals also had commensal bacteria inside their spleens, so it is possible that the shortened villi enabled translocation of the bacteria across the epithelial gut barrier, which normally restricts the entry of harmful substances by separating the intestinal organs from the external environment.

The spleens and brains of Hpb/WNV co-infected mice also contained markedly reduced numbers of WNV-specific CD8+ T cells, which usually contribute to the clearance of WNV infection. The authors reasoned that the lack of CD8+ T cells was likely due to bacterial invasion, which may impact the activation of key immune cell types involved in triggering the differentiation of virus-specific CD8+ T cells. Remarkably, treatment of Hpb/WNV co-infected mice with antibiotics reversed these kinetics, leading to increased levels of WNV-specific CD8+ T cells, a result that correlated with diminished bacterial levels in the blood and spleen. This observation suggested that bacterial invasion of lymphoid tissues is largely responsible for impaired WNV-specific CD8+ T cell responses during co-infection with Hpb and WNV.   

The clearance of helminth infections is typically mediated by the activation of “type 2” immune responses, which are characterized by the production of various cytokines, including IL-4. Binding of IL-4 to its cognate cell surface receptor activates STAT6, a cytoplasmic transcription factor that ultimately signals the activation of macrophages and differentiation of T cells. To determine if these type 2 immune responses contribute to the lethality observed after co-infection with Hpb and WNV, the authors performed the next set of experiments on “STAT6 null” mice, which lack the STAT6 gene. Interestingly, STAT6 null mice co-infected with Hpb and WNV did not have any of the increased lethality observed in wild type mice. Hpb/WNV-co-infected STAT6 null mice also had reduced overall viral levels and no gut abnormalities characteristic of enhanced pathogenesis, suggesting that STAT6 signaling is needed for development of enhanced disease.

To determine the relevance of IL-4 in the STAT6 signaling cascade, wild type mice were infected with WNV alone, followed by administration of IL-4. This regimen led to increased pathogenicity like that observed in Hpb/WNV co-infected mice, including decreased survival, weight loss, and higher WNV burden in the brain and gut. In contrast, the same WNV/IL-4 regimen given to STAT6 null mice did not produce the same pathogenicity, suggesting that the effects of IL-4 are dependent on the presence of STAT6.  

Type 2 immune responses in the gut are initiated by tuft cells, which are specialized cells of the intestinal epithelium that are capable of multiplying in response to the presence of certain parasites. To see whether tuft cells are needed for Hpb-mediated enhanced WNV disease, the authors infected “tuft null” mice (mice lacking tuft cells) with both Hpb and WNV. These mice did not develop any of the effects characteristic of enhanced pathogenesis, suggesting that tuft cells are needed for development of enhanced disease.

Tuft cells selectively express a receptor for succinate, a metabolite needed for energy production. To see whether succinate sensing is involved in enhanced WNV disease, wild type mice were administered succinate and were then infected with WNV. All these mice developed enhanced disease, even in the absence of Hpb infection. However, the same succinate/WNV regimen in tuft null mice, or in mice expressing tuft cells but lacking succinate receptors, did not lead to enhanced disease. These experiments further confirm that succinate receptor-expressing tuft cells are needed to initiate the signaling pathway leading to enhanced disease. Collectively, these results suggest that Hpb-mediated activation of type 2 immune responses in intestinal epithelial cells depends on tuft cells and might be initiated by succinate sensing.

Similar enhanced disease characteristics were also observed after co-infection of mice with Hpb and Zika virus or Powassan virus, suggesting that co-infection with helminths may enhance the disease severity of multiple human flaviviruses. Although these parasitic worm/flavivirus co-infections are currently more common in tropical regions, their prevalence may spread with increasing global climate changes/warming. This study outlines a fascinating aspect of the interface between parasitic and viral infections that warrants further exploration.

[For a more detailed discussion of this paper, I recommend This Week in Parasitism episode 194]

Early Immune Responses to Herpes Simplex Virus Type I Infection

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by Gertrud U. Rey

Herpes simplex viruses infect cells of the skin and mucous membranes, where they establish a lifelong persistent infection in sensory neurons. Sporadic reactivation and viral shedding may lead to painful oral and genital disease and a three to five-fold increased risk of HIV transmission. There is currently no vaccine to prevent infection with herpes simplex virus type 1 or type 2 (HSV-1 or HSV-2).

Until recently it was thought that initial interactions of HSV-1 with the immune system only involve Langerhans cells. Langerhans cells are skin-resident sentinel macrophages that detect microbial antigens, and they engulf, process, and present these antigens to T cells for downstream immune functions. However, a recent study suggests that early during infection, HSV-1 also interacts with a newly identified immune cell known as an epidermal conventional dendritic cell type 2 (Epi-cDC2). Like Langerhans cells, dendritic cells can swallow microbe-infected cells and present the microbial antigens to T helper cells, ultimately triggering the actions of cytotoxic T cells, which directly kill infected cells.

The study aimed to better define the role of Epi-cDC2s in early HSV-1 infection using ex vivo explants as a model system. The explants consisted of pieces of human inner foreskin that were mounted on specialized gelatin scaffolds to mimic the in vivo environment encountered by HSV-1 during infection. The authors exposed the explants to an HSV-1 virus in which a viral membrane protein was fused to a green fluorescent protein (GFP), allowing them to visually track a resulting infection using a fluorescence microscope. This method revealed that at 24 hours after exposure to the GFP-tagged HSV-1, both Langerhans cells and Epi-cDC2s contained the virus in their cytoplasm, suggesting that these cells either engulfed HSV-1-infected skin cells and/or were themselves infected by HSV-1.

To determine whether the presence of HSV-1 in the cytoplasm of Epi-cDC2s resulted from infection and replication and not just from engulfing infected skin cells, the authors first did the following. They exposed cell cultures of Epi-cDC2s to HSV-1. After six hours of this exposure, Epi-cDC2s contained about as much virus as did control Langerhans cells, which are known to be infected by HSV-1. However, at 18 hours, Epi-cDC2s contained significantly higher HSV-1 levels than Langerhans cells, suggesting increased entry/uptake of virus into Epi-cDC2s compared to Langerhans cells. Next, to assess whether HSV-1 was also replicating in the Epi-cDC2 cells, not just entering them, the authors treated the cells with a fluorescent antibody that binds ICP27, a viral protein needed for replication. A significantly greater portion of Epi-cDC2s than Langerhans cells expressed ICP27, suggesting that HSV-1 was replicating, and doing so more efficiently in Epi-cDC2s.

Viruses may enter a host cell by a variety of mechanisms. One common mechanism, called receptor-mediated endocytosis, involves the formation of cell membrane-derived vesicles. In one version of this process, which requires a low pH, viral binding to a cell surface receptor triggers the cellular membrane to fold inward and form a slightly acidic “endosome” around the virus. Another version of receptor-mediated endocytosis is not dependent on a low pH, but requires cholesterol molecules and the motor protein actin to form cell surface protrusions called “ruffles.” When the ruffles become large enough, they collapse back onto the membrane and form large fluid-filled vesicles encasing the virus. In both of these versions of receptor-mediated endocytosis, the resulting vesicles enter the cytoplasm, where they eventually release their contents. In yet another mechanism of entry, also independent of acidic pH, viruses may simply fuse with the plasma membrane and deliver their contents into the cytoplasm.

Although HSV-1 can enter cells by any of these pathways, its entry mechanism differs in different types of cells. To determine which pathway HSV-1 uses to enter Langerhans cells and Epi-cDC2s, the authors treated both types of cells with a drug that prevents acidification of endosomes. They then infected the cells with the GFP-tagged HSV-1 and measured infection by quantitating GFP with a fluorescence microscope. Increasing doses of the drug led to increased inhibition of infection of Langerhans cells, suggesting that these cells are infected with HSV-1 via a pH-dependent mechanism. In contrast, the drug did not affect infection of Epi-cDC2s, suggesting that HSV-1 does not require an acidic pH for entering Epi-cDC2s.

To determine whether HSV-1 entry into Epi-cDC2s occurred via actin and cholesterol-dependent endocytosis, the authors treated Epi-cDC2s with inhibitors of actin or cholesterol prior to infection. Both treatments led to significant reduction in GFP fluorescence inside the cells, suggesting that cholesterol and actin are both important mediators of HSV-1 entry into Epi-cDC2s.

Langerhans cells express a cell surface receptor called langerin, which mediates entry of HIV and influenza A. To see whether this receptor is also required for entry of HSV-1, the authors infected Langerhans cells with HSV-1 in the presence of an antibody that neutralizes langerin. This inhibition of langerin expression led to diminished infection of Langerhans cells, suggesting that langerin is required for HSV-1 entry into them. In contrast, inhibition of langerin on Epi-cDC2s had no effect on HSV-1 infection efficiency, suggesting that, even though Epi-cDC2s do express some langerin, this receptor is not required for HSV-1 entry of these cells.

HSV-1 and HSV-2 are of high public health concern, and a vaccine to prevent infection with these viruses is urgently needed. Immune control of HSV-1/-2 infection and resolution of genital herpes lesions requires the collective action of various types of T cells, which are likely primed by different dendritic cell subsets. Understanding the dynamics of the initial interactions of HSV-1 and HSV-2 with cells of the immune system may result in better strategies for HSV-1/-2 vaccines. The pathways described here have important implications in vaccine design and prevention of persistent infection of neuronal cells.

Are the SARS-CoV-2 Vaccines Safe for Pregnant and Lactating People?

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by Gertrud U. Rey

Vaccination is the gold standard for preventing infectious diseases and reducing the impact of emerging pathogens. As more and more people are becoming immunized against SARS-CoV-2, a prominent question continues to arise: are the vaccines safe for pregnant and breast-feeding people?

(Image credit: Shutterstock)

None of the clinical trials for SARS-CoV-2 vaccines included pregnant or lactating subjects. Pregnant people in particular are a historically difficult group to study in the context of drug safety because of concerns of liability over potential adverse effects of a new product on a fetus. This lack of safety data further complicates treatment of SARS-CoV-2 infection in pregnant people, who already have an increased risk for premature birth and miscarriage if they become infected. Based on these risk factors, the currently available data, and interim recommendations by the Advisory Committee on Immunization Practices for people over the age of 16, the American College of Obstetricians and Gynecologists recommends that pregnant and lactating people should have access to SARS-CoV-2 vaccines. Fortunately, many pregnant and lactating people have opted to be vaccinated, and the data on the safety and efficacy of vaccination in these two cohorts are beginning to emerge.

A recent publication in the American Journal of Obstetrics and Gynecology describes the safety and immunogenicity of SARS-CoV-2 vaccines in pregnant and lactating women compared to non-pregnant controls and women who had been naturally infected with SARS-CoV-2 during pregnancy. The researchers collected blood and breast milk from 131 women of reproductive age, including 84 pregnant, 31 lactating, and 16 non-pregnant participants who had received two doses of either the Pfizer/BioNTech or Moderna mRNA COVID-19 vaccine; and analyzed the samples for the presence of IgG and IgA antibodies. IgG antibodies are mostly present in the blood and provide the majority of antibody-based immunity against invading pathogens. IgA antibodies are predominantly found in mucus membranes and the fluids secreted by these membranes, including saliva, tears, and breast milk. IgA antibodies are particularly suitable for protecting against pathogens like SARS-CoV-2, which invade the body through these membranes.

Samples were collected at the time of the first and second vaccination, 2 to 6 weeks after the second dose, and at delivery. The researchers also collected umbilical cord blood from 10 infants born during the study period.

The main findings were as follows. Side effects resulting from vaccination were rare and occurred with similar frequency in all participants. These findings are consistent with post-vaccination data tracked by the CDC via the V-safe application, suggesting that post-vaccination reactions in pregnant people are not significantly different from those in non-pregnant people.

Pregnant, lactating, and non-pregnant vaccine recipients produced significantly higher levels of SARS-CoV-2-specific IgG than pregnant women who had been naturally infected. Interestingly, the second vaccine dose led to increased concentrations of coronavirus-specific IgG but not IgA, in both maternal blood and breast milk. The authors speculate that this lack of IgA boosting may have to do with the intramuscular route of immunization, which usually does not induce very high levels of serum IgA. The role of IgA in the serum is mostly secondary to IgG, in that IgA mediates elimination of pathogens that have breached the mucosal surface. In contrast, natural infection induces higher levels of mucosal (IgA) antibodies because SARS-CoV-2 enters the body through the mucosal surfaces. Nevertheless, studies have shown that IgG in breast milk is critical for protecting newborns from other pathogens like HIV, RSV, and influenza. It is possible that this type of antibody is equally important for protecting infants from SARS-CoV-2.

It was previously unclear whether vaccine-induced SARS-CoV-2 antibodies are transferred across the placenta to the fetus. However, the authors also found vaccine-induced IgG in cord blood, suggesting that these antibodies do cross the placenta, as has been previously observed after vaccination of pregnant people against influenza virus, Bordetella pertussis, and other pathogens. Whether these antibodies protect the baby from SARS-CoV-2 infection after birth, remains to be determined.

This study provides the first set of clinical data suggesting that vaccination of pregnant and lactating people against SARS-CoV-2 is both safe and beneficial. Some people have expressed concern over the possible incidence of fever following the second vaccine dose in pregnant people; however, the potential risk to the fetus from a 24-hour fever must be weighed against the possibility of chronic, severe COVID-19 in the mother. Accumulating data suggest that the benefits of vaccination during pregnancy and lactation outweigh the risks of adverse effects to the fetus or infant and align with the abundance of clinical data showing the beneficial effects of vaccinating these groups of people against influenza virus. Analyses of cord blood from infants whose mothers were naturally infected with SARS-CoV-2 during pregnancy indicate that the potential for transfer of antibodies is greater early during gestation, suggesting that immunization during the earlier stages of pregnancy might be preferable. However, more studies are needed to assess the optimal timing of maternal vaccination to achieve enhanced neonatal immunity. In the meantime, these preliminary data allow pregnant and lactating people to make more informed decisions, and also aid physicians in providing evidence-based recommendations.

One and Done

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by Gertrud U. Rey

On February 27, 2021, the FDA issued an emergency use authorization for a third SARS-CoV-2 vaccine. The vaccine was developed by Janssen Pharmaceutica, a Belgium-based division of Johnson & Johnson, in collaboration with Beth Israel Deaconess Medical Center in Boston. Perhaps the most exciting feature of this new vaccine is that it only requires one dose to be effective in inducing an immune response.

The vaccine is named Ad26.COV2.S because it consists of a human adenovirus vector, with a DNA genome, into which has been inserted the gene that encodes the full-length SARS-CoV-2 spike protein (pictured). Ad26.COV2.S is similar to AstraZeneca’s vaccine, based on a different adenovirus, and with a slightly different version of spike, which is not yet authorized in the U.S. The notion of using a virus as a vector to deliver vaccines to humans is based on the ability of viruses to enter cells by attaching to host cell receptors and releasing their genome into the cell. Upon injection into a vaccine recipient, the vaccine vector should enter cells and serve as a code for host proteins to synthesize the SARS-CoV-2 spike protein from the inserted gene. Ideally, the spike protein will then act as an antigen to prime the immune system to recognize SARS-CoV-2 if it infects the body at a later time. 

Adenoviruses are particularly suitable as vectors for delivering foreign genes into cells because they have a double-stranded DNA genome that can accommodate relatively large segments of foreign DNA, and because they infect most cell types without integrating into the host genome. However, because of the prevalence of adenovirus infections in humans, most people have adenovirus-specific antibodies that could bind and neutralize these vectors, thus rendering them less effective at stimulating antibodies to the inserted gene product. AstraZeneca circumvented this issue by using an adenovirus of chimpanzee origin that does not normally infect humans. The adenovirus used to make Ad26.COV2.S (Adenovirus 26) is of human origin; however, when tested, most people have very few antibodies that inactivate this adenovirus, compared to antibodies against other adenoviruses. Thus, potential Ad26.COV2.S recipients are less likely to have pre-existing antibodies to the adenovirus vector itself. To optimize Adenovirus 26 for use as a vaccine vector, Janssen investigators deleted the gene that regulates viral replication, thus ensuring that the virus vector cannot cause an infection in human cells.

During infection, the SARS-CoV-2 viral particle fuses with the host cell membrane; a process that is mediated by two main events: 1) a structural rearrangement of the spike protein from its pre-fusion conformation; and, 2) cleavage of the spike protein by a cellular enzyme called furin. Based on the knowledge that the pre-fusion, uncleaved form of spike is more stable and immunogenic, Janssen investigators also inserted two mutations into the spike gene: one that locks the translated spike protein into its pre-fusion conformation, and one that prevents its cleavage by furin.  

The FDA’s decision to issue an emergency use authorization for Ad26.COV2.S was based on safety and efficacy data from an ongoing Phase III clinical trial done in 39,321 participants who received either a single dose of Ad26.COV2.S or a placebo control. The trial was randomized, meaning that participants were randomly assigned to the experimental group receiving the Ad26.COV2.S vaccine, or the control group, so that the only expected differences between the experimental and control groups were the outcome variables studied (safety and efficacy). Randomizing trial participants eliminates unwanted effects that have nothing to do with the variables being analyzed. The trial was also double-blinded, meaning that neither the investigators nor the subjects knew who was receiving a particular treatment. Double-blinding leads to more authentic conclusions because they reduce researcher bias.

The basic findings of the trial were as follows:

  • side effects related to vaccination were mild to moderate; and
  • the vaccine was
    • 66% effective at preventing moderate to severe COVID-19 across all geographic areas and age groups (U.S., South Africa, and six countries in Latin America);
    • 72% effective at preventing moderate to severe COVID-19 across all age groups in the U.S.; 
    • 85% effective at preventing severe disease; and
    • 100% effective at preventing COVID-19-related hospitalization and death as of day 28 after vaccination.

The apparently reduced efficacy of Ad26.COV2.S compared to the Moderna and Pfizer vaccines has led to considerable public skepticism. However, this is an unfair comparison for several reasons. Ad26.COV2.S was tested at a time when more variants were in circulation, including in places where the Moderna/Pfizer vaccines are thought to be less effective against locally circulating variants. Some limited data also suggest that Ad26.COV2.S might protect from asymptomatic infection and may thus prevent transmission from vaccinated individuals to non-vaccinated individuals. Although there is some evidence to suggest that the Pfizer vaccine has a similar effect, no such data exist yet for the Moderna vaccine.

The most critical measure of a vaccine’s efficacy is how well it prevents severe disease, hospitalizations, and deaths, and in this regard, all three vaccines are comparable. Moreover, Ad26.COV2.S has at least two advantages over the Pfizer/Moderna vaccines: 1) it does not require a freezer and can be stored in a refrigerator for up to three months; and, 2) it can be administered in a single dose. This will increase vaccine uptake, because people won’t have to get two shots and/or remember to get the second shot.  It also makes it easier to immunize people with limited access to healthcare, such as the homeless and people living in remote areas. When all these factors are considered together, it is clear that Ad26.COV2.S will be a crucial additional tool in the fight against this pandemic.

Bacteria Make Antivirals

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by Gertrud U. Rey

Antiviral agents are a class of drugs that inhibit the replication of viruses at various stages of their replication cycle. One class of antivirals is small molecules known as “chain terminators,” because their addition to the end of a chain of DNA or RNA prevents the addition of any subsequent nucleotides, thus ending the replication of DNA or RNA.

Individual RNA nucleotides consist of a ribose sugar that is connected to one of four different bases (adenine, guanine, cytosine, or uracil) via its 1′ carbon and a group of three phosphates via its 5′ carbon. For example, the ribose in the nucleotide cytidine triphosphate (CTP) is connected to a triphosphate group and a cytosine base (left side of Figure). Addition of this CTP to the end of a replicating RNA strand results in loss of two of its phosphates and connection of the remaining phosphate to the 3′ hydroxyl (OH) of the ribose in the preceding nucleotide in the strand. The absence of a hydroxyl at the 3′ carbon of a nucleotide ribose (right side of Figure) would prevent the binding of a subsequent nucleotide and would thus make that nucleotide the last one in the chain.  

Viral infection in humans induces production of interferon, which further stimulates the expression of other genes, one of which is viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon inducible). Research in recent years has shown that viperin catalyzes the conversion of CTP to 3′-deoxy-3′,4′-didehydro(ddh)-cytidine triphosphate (ddhCTP) (right side of Figure). As the “deoxy” portion of its name implies, ddhCTP lacks a hydroxyl at the 3′ carbon of its ribose sugar, making it a chain terminator. In human cells, the conversion of CTP to ddhCTP by viperin leads to broad antiviral activity against various viruses, including human cytomegaloviruses, West Nile virus, dengue virus, hepatitis C virus, and HIV, without any toxic effects on host DNA replication.

Some bacteria and archaea encode genes with notable sequence similarity to vertebrate viperins; however, until recently, their role was unclear. Because of the antiviral role of viperins in human cells, the authors of a recent publication aimed to determine whether prokaryotic viperins participate in defenses against viruses that infect bacteria, known as bacteriophages (or, simply “phages”). Knowing that genes encoding antiviral defense proteins tend to cluster together in the prokaryotic genome, the authors searched for the presence of such clusters in a variety of bacterial and archaeal genomes. They found that although most clusters of viperin-like genes did not colocalize with defense genes, 60% of the genes in one cluster  were located near genes encoding CRISPR-Cas systems, restriction enzymes, and other bacterial defense genes.   

To see whether the prokaryotic viperin-like genes in these clusters encoded proteins with antiviral activity (pVips), the authors cloned 59 of these pVip genes into E. coli under the control of an inducible promoter, allowing their expression to be switched on or off. Infection of these pVip-producing E. coli with a range of phages revealed that about half of the tested pVips had inhibitory activity against the phages. Most of the pVips inhibited bacteriophage T7, but some of them also inhibited P1, lambda, SECphi6 and SECphi18 phages, without any toxic effects on the bacterial host cells. Surprisingly, when human viperin was expressed in E. coli under the same conditions, it protected against infection by T7 phages, suggesting that the antiviral functions of viperin and viperin-like gene products are highly conserved.

The authors next wanted to determine whether pVips catalyze production of ddhCTP, like human viperins. Assuming that small molecule fragments extracted from pVip-producing E. coli having anti-phage activity would include modified nucleotides, the authors analyzed E. coli lysates by mass spectrometry, a technique that reveals the chemical identity of molecules and other chemical compounds. They then compared the small molecules identified from cells producing pVips to molecules obtained from E. coli containing human viperin. This analysis revealed that cells producing some prokaryotic viperin-like molecules contained derivatives of CTP, including ddhCTP, while others did not, suggesting that only some viperins catalyze the production of ddhCTP. However, some prokaryotic viperins seemed to produce modified ddh derivatives of other nucleotides, like GTP and UTP. Collectively, these results suggest that prokaryotic viperins produce antiviral nucleotides, just like human viperins.

Although environmental bacteria have long been used to identify novel antibiotics, the notion of extracting antivirals from bacteria is new. Nevertheless, many of the antiviral drugs used in clinical settings are chain terminators, including acyclovir for herpes virus, AZT for HIV, and sofosbuvir for treating hepatitis C virus infections. The present study may have revealed an additional source of diverse antiviral molecules that could be adapted for clinical treatment of human viral infections.

[The study described in this article was also discussed at length on TWiM 233.]