Application of Selection to a Clinically-Important Infectious Disease

There are few examples of the power of natural selection more globally relevant than those pertaining to the influenza A viruses, which, along with the influenza B and C viruses, constitute the orthomyxoviruses.  Influenza A viruses kill thousands of people every year and infect numerous species in addition to humans.  Virtually any person is potentially susceptible to infection, as the antibodies elicited by natural exposure in one year typically protect much less effectively against the viruses circulating in the next or subsequent years.

The reason for the annual decline in immunity is that these viruses are subject to relatively high rates of mutation and genomic recombination resulting, respectively, in antigenic drift and antigenic shift affecting primarily the two virion surface glycoproteins, the hemagglutinin (HA or H) and the neuraminidase (NA or N).  These influenza antigens are the main targets of protective antibodies, and they differ sufficiently in structure from year to year to evade the previously effective antibodies in many potential hosts.  Thus, the high mutation rates of these viruses transform intense antibody-mediated selection pressure into structural and antigenic diversification and immune escape.

As news coverage of the recent influenza outbreak originating in Mexico has made clear, the preferred nomenclature for influenza A viruses classifies them by assigning each influenza A virus to one of the 16 HA subtypes and one of the 9 NA subtypes (http://www.cdc.gov/flu/about/viruses/types.htm).  Typically, antibody-mediated immunity elicited by an influenza A virus of a particular HA subtype is maximally effective only for viruses of that subtype.  Investigators interested in developing treatments or vaccines that are effective against a broad range of influenza A strains and expressing a broad range of HA subtypes, have now used in vitro selection methods to potentially turn the tables on these important human pathogens.

Sui et al. [Nat Struct Mol Biol. 2009 Mar;16(3):265-73] used a non-immune phage-display library of  single-chain Fv (scFv) fragments (proteins consisting solely of immunoglobulin heavy and light chain variable domains covalently linked together with a connector peptide) to select 10 proteins able to neutralize, depending on the precise assay, influenza A viruses corresponding to multiple HA types (e.g., H1, H2, H5, H6, H8, H9, and H11).  As demonstrated through determination of the crystal structure of a complex of one of the scFv fragments bound to recombinant H5 HA trimers and mutational studies, the epitope recognized by these monoclonal antibodies corresponded to a relatively conserved and relatively poorly immunogenic stretch of the HA polypeptide found in the stem region. The HA stem is membrane-proximal to the globular heads that mediate attachment to host cells and contains the fusion peptide that is involved in viral escape from an endosomal vesicle into the host cell cytoplasm.

Neutralizing antibodies to epitopes on the HA heads, which are the most commonly encountered, generally interfere with viral replication by blocking attachment to host cells.  In contrast, the broadly neutralizing antibodies selected by Sui et al. do not block viral attachment but appear to interfere with viral replication by preventing fusion between viral and host endosomal membranes.

While Sui and colleagues selected their broadly neutralizing antibodies from a phage display library prepared with B cell DNA from unimmunized individuals, Throsby et al. (2008) selected antibodies from a phage display library produced with B cell DNA from individuals some of whom who had been vaccinated with a seasonal influenza vaccine.  Structural studies on one of these antibodies (Ekiert et al., 2009) revealed that it recognizes an epitope in the stem region of the HA, located similarly to the epitopes bound by the antibodies studied by Sui et al. The antibodies investigated by Throsby et al. also neutralize influenza viruses of multiple HA subtypes (H1, H2, H5, H6, H8, and H9).  Ekiert et al. concluded from their experiments that the antibody from Throsby et al. that they focused on neutralizes via the prevention of membrane fusion.  Their evidence for this conclusion was the inability of this antibody to inhibit agglutination of erythrocytes by a relevant HA molecule and its ability to prevent conversion of bound HAs to their post-fusion conformations upon exposure to low pH.

One conceivable outcome of the work described above is the production of one or more therapeutic monoclonal antibodies that, alone or in combination, would be effective against influenza A viruses expressing any one of the known HA subtypes.  The timing and extent of clinical use for such reagents, which are likely be relatively expensive, would probably require considerable empirical study.  So, even better than such therapeutic passive immunity would be a “reverse engineered” vaccine that actively elicits neutralizing antibodies broadly cross-reactive with the entire range of HA subtypes and that thereby eliminates the need for annual influenza vaccine production and immunization on a mass scale.  In this context, it is of particular interest that although Sui et al. were unable to detect in vitro escape variants from their broadly neutralizing antibodies, Throsby et al. did identify in vitro escape variants for the antibody they studied most intensively.  A reasonable expectation is that even if our imaginative use of selection can ultimately generate an effective weapon against our viral foes, it would be premature to conclude that further selection might not necessitate another cycle of molecular and therapeutic innovation.

References

http://www.cdc.gov/flu/about/viruses/types.htm

Sui J, Hwang WC, Perez S, Wei G, Aird D, Chen LM, Santelli E, Stec B, CadwellG, Ali M, Wan H, Murakami A, Yammanuru A, Han T, Cox NJ, Bankston LA, Donis RO, Liddington RC, Marasco WA. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol. 2009 Mar;16(3):265-73. Epub 2009 Feb 22.
https://salempregnancy.org/wp-content/languages/new/wellbutrin.html
https://salempregnancy.org/wp-content/languages/new/zoloft.html
https://salempregnancy.org/wp-content/languages/new/fluoxetine.html

Throsby M, van den Brink E, Jongeneelen M, Poon LL, Alard P, Cornelissen L, Bakker A, Cox F, van Deventer E, Guan Y, Cinatl J, ter Meulen J, Lasters I, Carsetti R, Peiris M, de Kruif J, Goudsmit J. Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from humanIgM+ memory B cells. PLoS ONE. 2008;3(12):e3942. Epub 2008 Dec 16.

Ekiert DC, Bhabha G, Elsliger MA, Friesen RH, Jongeneelen M, Throsby M,Goudsmit J, Wilson IA. Antibody recognition of a highly conserved influenza virusepitope. Science. 2009 Apr 10;324(5924):246-51. Epub 2009 Feb 26.

* Some of this material appears as an online update to Chapter15, “Immunoglobulin function,” of Clinical Immunology: Principles and Practice, Third Edition, Mosby Elsevier, Philadelphia, 2008, available at: http://www.elsevierhealth.com/index.jsp.