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About viruses and viral disease

hemagglutinin

Universal influenza vaccines

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The need to re-formulate the influenza virus vaccine in response to viral antigenic drift and shift makes for complex logistics of vaccine production and administration. Surveillance programs must be conducted each year to identify strains that are likely to predominate and cause disease. Wouldn’t it be simpler if a single vaccine could be developed that would confer protection against a broad range of viral strains? Results from the past year suggest that such a vaccine might be closer than previously thought.

The influenza viral HA protein consists of a globular head atop a stem that is embedded in the virion membrane (figure). Most protective antibodies are directed against the head of the HA molecule. Rare antibodies that block infection with a broad range of influenza virus strains are directed toward the conserved stalk of the viral surface glycoprotein HA. This observation was taken a step further by showing that sequential immunization with different viral HAs, or with HA lacking the globular head, induce broadly neutralizing antibodies. Peter Palese discussed these approaches on TWiV #102.

In another approach, neutralizing antibodies have been induced by immunizing first with plasmid DNA, followed by a boost with recombinant adenovirus encoding the HA protein. Mice were immunized first with plasmid DNA encoding an H1 HA from the 2006-2007 influenza season, then boosted with a recombinant adenovirus encoding the same HA protein. Sera from immunized mice neutralized strains of H1N1 influenza virus dating to 1934, as well as H2N2 and H5N1 viruses. When inoculated with a 1934 H1N1 virus, immunized mice were protected from lethal disease. Immunization of ferrets with a similar regimen also protected these animals from lethal disease. Broadly neutralizing antibodies were elicited in nonhuman primates by this prime-boost regimen.

Both the plasmid DNA and the recombinant adenovirus encoded the full-length HA protein, with both the globular head and fibrous stem. However, the broadly neutralizing and protective antibodies were directed against the stem. Anti-HA stem antibodies were also identified in monkeys that had been immunized with the prime-boost combination.

Why doesn’t the seasonal influenza vaccine elicit broadly neutralizing antibodies? These vaccines induce antibodies that almost exclusively bind the variable head of the HA, not the conserved stem. The reason probably lies in how the vaccines are prepared: virions are inactivated by treatment with detergent and formaldehyde, a process that destroys the particle. Consequently, the vaccine contains mainly HA and NA and not other components that can help shape a more diverse antibody repertoire. In contrast, it is known that plasmid-based priming can stimulate B cells to produce a more diverse set of antibodies.

The strategy of priming with plasmid DNA followed by boosting with recombinant adenovirus will likely be evaluated in clinical trials for the ability to protect against natural infection with influenza virus. The possibility of a broadly protective influenza virus vaccine that would be taken perhaps every 10-20 years is rapidly becoming a reality.

Wang TT, Tan GS, Hai R, Pica N, Petersen E, Moran TM, & Palese P (2010). Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS pathogens, 6 (2) PMID: 20195520

Wei CJ, Boyington JC, McTamney PM, Kong WP, Pearce MB, Xu L, Andersen H, Rao S, Tumpey TM, Yang ZY, & Nabel GJ (2010). Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science (New York, N.Y.), 329 (5995), 1060-4 PMID: 20647428

Virology pop quiz

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BaculovirusThis week’s pop quiz involves analysis of an AFP news article entitled “US company makes first batch of swine flu vaccine“. The article reports that Protein Sciences has been awarded a contract from the US Department of Health and Human Services to produce a vaccine by synthesizing the viral HA protein in insect cells. Here are two paragraphs from the article:

They warned that the virus could mutate during the southern hemisphere’s flu season before returning north in a more lethal form in autumn, in a pattern similar to that seen in the deadly 1918 flu pandemic, which claimed an estimated 20 to 50 million lives around the globe.

The CDC (Centers for Disease Control and Prevention) sent us a dead virus, which is perfectly safe, and then we extracted genetic information from that virus.

What is wrong with these statements? Post your answers in the comments section.

For extra credit, critique this statement from the same article:

Protein Sciences’ technology is also safer “because these caterpillars don’t have any association with man or other animals, so there’s no chance for their cells to learn how to propagate human viruses,” Adams told AFP.

Influenza HA cleavage is required for infectivity

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The influenza virus hemagglutinin (HA) is the viral protein that attaches to cell receptors. The HA also plays an important role in the release of the viral RNA into the cell, by causing fusion of viral and cellular membranes. HA must be cleaved by cellular proteases to be active as a fusion protein.

The HA on the influenza virion is a trimer: it is made up of three copies of the HA polypeptide. The cleavage site for cell proteases on the HA protein is located near the viral membrane.

influenza-ha-cleavage

In the diagram, the globular head of the HA protein, which attaches to cell receptors, is at the top, and the viral membrane is at the bottom. For clarity, only one HA cleavage site is labeled. The uncleaved form of the protein is called HA0; after cleavage by a cellular enzyme, two proteins are produced, called HA1 (blue) and HA2 (red). The two subunits remain together at the surface of the virus particle. The new amino(N)-terminal end of HA2 that is produced by cleavage contains a sequence of hydrophobic amino acids called a fusion peptide. During entry of influenza virus into cells, the fusion peptide inserts into the endosomal membrane and causes fusion of the viral and cell membranes. Consequently, the influenza viral RNAs can enter the cytoplasm. The fusion process is described in a previous post.

If the HA protein is not cleaved to form HA1 and HA2, fusion cannot occur. Therefore influenza viruses with uncleaved HA are not infectious. Cleavage of the viral HA occurs after newly synthesized virions are released from cells. Influenza viruses replicate efficiently in eggs because of the presence of a protease in allantoic fluid that can cleave HA. However, replication of many influenza virus strains in cell cultures requires addition of the appropriate protease (often trypsin) to the medium.

In humans, influenza virus replication is restricted to the respiratory tract, because that is the only location where the protease that cleaves HA is produced. However, the HA protein of highly pathogenic H5 and H7 avian influenza virus strains can be cleaved by proteases that are produced in many different tissues. As a result, these viruses can replicate in many organs of the bird, including the spleen, liver, lungs, kidneys, and brain. This property may explain the ability of avian H5N1 influenza virus strains to replicate outside of the human respiratory tract.

Like the HA proteins of highly pathogenic H5 and H7 viruses, the HA of the 1918 influenza virus strain can also be cleaved by ubiquitous cellular proteases. Consequently, the virus can replicate in cell cultures in the absence of added trypsin.

The H5 and H7 HA proteins have multiple basic amino acid residues at the HA1-HA2 cleavage site which allows cleavage by widely expressed proteases. But the 1918 H1 HA does not have this feature. Nor does the 1918 N1 help recruit proteases that cleave the HA, a mechanism that allows the A/WSN/33 influenza virus strain to multiply in cells without trypsin. An understanding of how the 1918 H1 HA protein can be cleaved by ubiquitous proteases is essential for understanding the high pathogenicity of this strain.

Chaipan, C., Kobasa, D., Bertram, S., Glowacka, I., Steffen, I., Solomon Tsegaye, T., Takeda, M., Bugge, T., Kim, S., Park, Y., Marzi, A., & Pohlmann, S. (2009). Proteolytic Activation of the 1918 Influenza Virus Hemagglutinin Journal of Virology, 83 (7), 3200-3211 DOI: 10.1128/JVI.02205-08