The results of recent structural studies have given us the ability to display the structure of Zika virus and of the viral E protein bound to antibody. In this video from Virus Watch I explain how the Zika virus particle is built, and how it interacts with an antibody that blocks infection, in beautiful three dimensional imagery.
Preprint servers, the structure of an antibody bound to Zika virus, blocking Zika virus replication in mosquitoes with Wolbachia, and killing carp in Australia with a herpesvirus are topics of episode #388 of the science show This Week in Virology, hosted by Vincent, Dickson, Alan, and Kathy.
You can find TWiV #388 at microbe.tv/twiv, or listen below.
You can find TWiV #387 at microbe.tv/twiv, or listen below.
On episode #345 of the science show This Week in Virology, the TWiVonauts review how the weather affects West Nile virus disease in the US, benefit of B cell depletion for ME/CFS patients, and an autoimmune reaction induced by influenza virus vaccine that leads to narcolepsy.
You can find TWiV #345 at www.microbe.tv/twiv.
Did you ever wonder what different virus infections you have had in your lifetime? Now you can find out with just a drop of your blood and about $25.
Immune defense systems of many hosts produce antibodies in response to virus infections. These large proteins, which are generally virus specific, can block or inhibit virus infection, and persist at low levels for many years after the initial infection. Hence it is possible to determine whether an individual has had a virus infection by looking for anti-viral antibodies in the blood. Up to now the process of identifying such antibodies has been slow and limited to one or a few viruses. A new assay called VirScan allows unbiased searches for all the virus antibodies in your blood, providing a picture of all your past infections.
To identify the human antivirome, DNAs were synthesized encoding proteins from all viruses known to infect humans – 206 species and over 1000 strains. These DNAs were inserted into the genome of a bacteriophage, so that upon infecting bacteria, the viral peptides are displayed on the phage capsid. These ‘display’ phages were then mixed with human serum, and those that were bound by antibodies were isolated. The DNA sequence of the phage genomes were then determined to identify the human virus bound by the antibodies.
This method was used to assay samples from 569 humans. The results show that each person had been exposed to an average of 10 viruses, with a range from a few to over 20 (two individuals had antibodies to 84 different virus species!). The most frequently identified viruses included herpesviruses, rhinoviruses, adenoviruses, influenza viruses, respiratory syncytial virus, and enteroviruses. The overall winner, found in 88% of samples, is Epstein-Barr virus.
These results are not unexpected: all of us are infected with at least a dozen viruses at any time, and the viruses identified in this study known to infect much of the human population. What was surprising is the absence of some common viruses, such as rotaviruses, and the ubiquitous polyomaviruses. According to serological surveys, the most common human viruses are the small, single-stranded DNA containing anelloviruses. Yet the related torque teno virus was only found in 1.7% of samples. These differences are likely due to a combination of technical and biological issues (e.g., failure of antibodies to certain viruses to persist in serum).
This new assay may one day become a routine diagnostic tool that is used along with complete blood counts and chemistries to know if a patient’s signs and symptoms might be attributable to a past virus infection. VirScan technology is not limited to virus infections – it can be used to provide a history of bouts with bacteria, fungi, and parasites.
VirScan might also allow us to determine which virus infections are beneficial, and which contribute to chronic diseases such as autoimmune or neurodevelopmental disorders or cancer. The assay can be used to conduct unbiased population-based studies of the prevalence of virus infections and their possible association with these diseases. Such connections were not previously possible with antibody assays that search for one virus at a time. This approach was not only inefficient, but required guessing the responsible virus.
Some other findings of this study are noteworthy. As expected, children had fewer virus infections than adults. HIV-positive individuals had antibodies to more viruses than HIV-negative individuals, also expected given the damage done by this virus to the immune system. Frequencies of anti-viral antibodies were higher outside of the United States, possible due to differences in genetics, sanitation, or population density. In most samples, there was a single dominant peptide per virus, although there were occasional differences among populations. This information might be useful for improving vaccines, or tailoring them to specific countries or regions.
Update: It would be very informative to use VirScan to search for antibodies against viruses that are not known to infect humans. Other animal viruses, plant viruses, insect viruses: to which do a significant fraction of humans respond? The information might identify other viruses that replicate in humans and which might constitute future threats (or present benefits).
ZMapp, a mixture of three antibodies against Ebola virus, became a household name after it was used to treat two Americans who were infected while working in Liberia. The structure of these antibodies bound to the Ebola virus glycoprotein suggest how they inhibit infection and ways to improve ZMapp.
The three monoclonal antibodies that comprise ZMapp (called c13C6, c2G4, and c4G7) were produced by immunizing mice with a recombinant vesicular stomatitis virus in which the glycoprotein was replaced with that from Ebola virus. Antibodies that bound the viral glycoprotein and protected mice from infection were identified, and three were made to resemble human antibodies and produced in tobacco plants. Ecco Zmapp!
Embedded in the membrane of the filamentous Ebola virus particle are many copies of the glycoprotein, seen as club-shaped spikes in the image to the right (image credit: ViralZone). The viral glycoprotein is essential for entry of the virus into cells. The antibodies in ZMapp are directed against the viral glycoprotein.
To determine how the antibodies bind the virus particle, they were individually mixed with purified Ebola virus glycoprotein, and the structures were determined by electron microscopy and image reconstruction. The results, shown in the illustration, indicate precisely where each antibody binds to the Ebola virus glycoprotein. The individual antibodies colored red (c2G4), yellow (c4G7), and purple (c13C6) are bound to a single Ebola virus glycoprotein in white, with the viral membrane below (Image credit).
The structures reveal that c13C6 (purple) binds at the tip of the viral glycoprotein, perpendicular to the plane of the viral membrane. The other two antibodies (red, yellow) bind at the base of the viral glycoprotein. Their binding sites overlap but are not identical (the Ebola virus glycoprotein is a trimer, and in the image, the yellow and red antibodies are shown binding to different subunits for clarity). Two other antibodies that block Ebola virus infection also bind at the base of the glycoprotein.
Antibody c13C6, which binds to the tip of the viral glycoprotein, does not neutralize viral infectivity. Nevertheless, it can protect animals from Ebola virus infection. This observation suggests that the c13C6 antibody may work in concert with complement, a collection of serum proteins, to block virus infection. It is not known why c13C6 antibody is non-neutralizing, but one possibility is that it binds to a part of the viral glycoprotein that is removed by an endosomal protease, cathepsin, before receptor binding in late endosomes.
Antibodies c2G4 and c4G7, which bind to the membrane-proximal part of the viral glycoprotein, neutralize viral infectivity. How they do so is not known, but one possibility is that they prevent structural changes of the viral protein that are essential for the fusion with the endosomal membrane, a process that delivers the viral nucleic acid into the cell cytoplasm.
These studies reveal two general areas of the Ebola virus glycoprotein that are important targets for antibodies that protect animals from Ebola virus infection. Those directed at the base of the glycoprotein neutralize infectivity while those that bind the tip do not. This information can now be used to isolate additional antibodies that bind either site. These can be used in animal protection studies to design mixtures that are even more potent than ZMapp.
Antigenic variation is a hallmark of influenza virus that allows the virus to evade host defenses. Consequently influenza vaccines need to be reformulated frequently to keep up with changing viruses. In contrast, antigenic variation is not a hallmark of poliovirus – the same poliovirus vaccines have been used for nearly 60 years to control infections by this virus. An exception is a poliovirus type 1 that caused a 2010 outbreak in the Republic of Congo.
The 2010 outbreak (445 paralytic cases) was unusual because the case fatality ratio of 47% was higher than typically observed (usually less than 10% of patients with confirmed disease die). The first clue that something was different in this outbreak was the finding that sera from some of the fatal cases failed to effectively block (neutralize) infection of cells by the strain of poliovirus isolated during this outbreak (the strain is called PV-RC2010). The same sera effectively neutralized the three Sabin vaccine viruses as well as wild type 1 polioviruses isolated from previous outbreaks. Therefore gaps in vaccination coverage were solely not responsible for this outbreak.
Examination of the nucleotide sequence of the genome of type I polioviruses isolated from 12 fatal cases revealed two amino acid changes within a site on surface of the viral capsid that is bound by neutralizing antibodies (illustration). The sequence of this site, called 2a, was changed from ser-ala-ala-leu to pro-ala-asp-leu. This particular combination of amino acid substitutions has never been seen before in poliovirus. Virus PV-RC2010, which also contains these two amino acid mutations, is completely resistant to neutralization with monoclonal antibodies that recognize antigenic site 2 (monoclonal antibodies recognize a single epitope, as opposed polyclonal antibodies which is a mixture of antibodies that recognize many epitopes. The antibodies in serum are typically polyclonal).
Poliovirus neutralization titers were determined using sera from Gabonese and German individuals who had been immunized with Sabin vaccine. These sera effectively neutralized the type I strain of Sabin poliovirus, as well as type 1 polioviruses isolated from recent outbreaks. However the sera had substantially lower neutralization activity against PV-RC2010. From 15-29% of these individuals would be considered not to be protected from infection with this strain.
Nucleotide sequence analysis of PV-RC2010 reveals that it is related to a poliovirus strain isolated in Angola in 2009, the year before the Republic of Congo outbreak. The Angolan virus had just one of the two amino acid changes in antigenic site 2a found in PV-RC2010.
It is possible that the relative resistance of the polioviruses to antibody neutralization might have been an important contributor to the high virulence observed during the Republic of Congo outbreak. The reduced ability of serum antibodies to neutralize virus would have lead to higher virus in the blood and a greater chance of entering the central nervous system. Another factor could also be that many of the cases of poliomyelitis were in adults, in which the disease is known to be more severe.
An important question is whether poliovirus strains such as PV-RC2010 pose a global threat. Typically the fitness of antigenically variant viruses is not the same as wild type, and therefore such viruses are not likely to spread in well immunized populations. Today some parts of the world have incomplete poliovirus immunization coverage, which together with the reduced circulation of wild type polioviruses leads to reduced population immunity. Such a situation could lead to the evolution of antigenic variants. This situation occurred in Finland in 1984, when an outbreak caused by type 3 poliovirus took place. The responsible strains were antigenic variants that evolved due to use of a sub-optimal poliovirus vaccine in that country.
The poliovirus outbreaks in the Republic of Congo and Finland were stopped by immunization with poliovirus vaccines, which boosted the population immunity. These experiences show that poliovirus antigenic variants such as PV-RC2010 will not cause outbreaks as long as we continue extensive immunization with poliovirus vaccines, coupled with environmental and clinical testing for the presence of such viruses.
On episode #257 of the science show This Week in Virology, the TWiV team consider how the kinase mTOR modulates the antibody response to provide broad protection against influenza virus, and explore the problems with scientific research.
You can find TWiV #257 at www.microbe.tv/twiv.
On episode #218 of the science show This Week in Virology, Vincent, Alan, and Welkin discuss how endogenous retroviruses in mice are held in check by the immune response.
You can find TWiV #218 at www.microbe.tv/twiv.
Welkin joins the TWiV team for a discussion of HIV prophlaxis using vectored antibodies, and the influenza H5N1 virus studies in ferrets that were not redacted.
Click the arrow above to play, or right-click to download TWiV 168 (59 MB .mp3, 98 minutes).
Links for this episode:
- Vectored HIV immunoprophylaxis (Nature)
- Vectored immunoprophylaxis – the Movie (YouTube)
- Adeno-associated virus (Wikipedia)
- In vitro evolution of H5N1 towards human receptor specificity (Virology)
- Endogenous viral genes non-essential in chicken (Nature)
- Rates of HIV transmission per coital act (J Inf Dis)
- TWiV on Facebook
- Letters read on TWiV 168
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