Contribution to the understanding of bird flu

January 2006 (still valid in 2008)


Contribution to the understanding of bird flu


History of a future “virtual” pandemic

In December 2003 the largest outbreak of highly pathogenic avian influenza H5N1 virus occurred among poultry in eight countries. In order to control this infection two hundred million poultry birds across Asia were destroyed and at the same time the H5N1 (avian variant) was isolated from a young girl who died after visiting her family in China.

Clinical cases have occurred in otherwise healthy young individuals (mortality rates approaching 70%), which were considered by specialists in epidemiology predictions as reminiscent of the 1918 Spanish influenza pandemic. No data were reported indicating if the victims of this infection were previously vaccinated one or several times against influenza, or regarding the time elapsed between the probable contact with an infected animal and the clinical signs, or the delay between the first signs evoking influenza and the admission at the hospital.

The populations exposed to influenza viruses or vaccinated recently bear antibodies recognizing the viral strains circulating in a certain territory and during a certain time and in people immunological “naïve” to new viral variants (absence of antibodies) the pathogenic power of these new viruses is objectively unpredictable. However, no data obtained from animal models or from theoretical predictions are able to predict the morbidity and mortality that may or may be not induce the new variant H5N1 in people vaccinated in 2004 and 2005.

The avian influenza A viruses are the result of the combination of twenty-four different proteins on their surface (15 Hemagglutinins and 9 Neuraminidases) and can be spread by healthy or sick migratory birds and waterfowl, carrying them in their intestines.

Before the event reported in 2003, the subtypes of influenza A virus mostly found in humans were H1 and H3 (H refers to the type of Hemagglutinin and N to the type of Neuraminidase, that are proteins expressed independently by the influenza viruses). The H and N are protein spices anchored on the viral surface at a ratio of about 8 to 1 and are capable of stimulating protective immune responses against infection (neo-synthesis of antibodies).

The influenza viruses continue to experience what is called “antigenic drift” by expressing modifications in the proteins expressed on their surface. These modifications allow them to escape partially or totally to the neutralization of antibodies induced by other surface proteins like those used as antigens for vaccines or those present in the virus that may have provoked previous infections. The continual viral modifications are not inducible by changes in the ecosystem or by any kind of “viral intelligence” or “viral intentionality” perpetrated to perpetuate information but are the consequence of the natural activity of all the enzymes replicating genes in biologic systems. Therefore, the biological machineries should be understood as bioreactors and not as photocopiers and the viral variability is a continuous natural process due to a lack of effective proofreading of the genes by the RNA polymerase, which result in the amino acid substitutions in surface antigens. The variations expressed on the viral surface may attain a threshold of differentiation with other viral-quasi-species. If this difference is located in the antibody-binding sites, the new viral generations may evade (at unpredictable degrees) immunity and re-infect immunised individuals.

The genes coding the information for influenza viruses are not structured as a continuum quantum of linear information but in segments physically separated. Because genetic information is segmented, when two different influenza viruses simultaneously infect cells, reassortments may occur and generate different hybrids that may not be properly neutralized by antibodies directed to the parents. The result of the mixture of different viral information called “antigenic shift” generates the new variants and its detection is the ground for the warning of the new pandemic.

Before 1997 the direct transmission of avian Influenza viruses to the human was considered practically impossible and it was thought that bird viruses did not bind directly to human receptors. A “virology dogma” established that intermediate hosts like the pigs (which carry both receptors) were necessary to allow the “mixing and reassortment” of viral genes to generate new strains capable to infect humans. However, the recent outbreak in poultry has spread a highly pathogenic virus from chicken to humans and has changed the understanding of the genesis of this yet “virtual” pandemic. It is now accepted that birds can shed reassorted pathogenic viruses to pigs, tigers, cats and humans.

The geographical expansion of the new H5N1 variant across Asia in an unprecedented event of transmission of viruses to humans and proof that a new avian-human reassortment did emerge and has been transmitted. The emergent virus is a necessary but not sufficient condition to predict sickness and death in humans. Figures predicted by Experts need a comprehensive revision, and diluted into realistic relativism: in the previous influenza pandemic, the coverage of the populations with influenza vaccines was much lower than at present. Moreover, during the pandemic occurred at the beginning of the 20th century and in the late 50s, the choice of agents to treat efficaciously bronco bacterial infections was limited and the management of inflammation, oedema, pyrexia, artificial ventilation and blood clotting were far from what is currently available today.

Today, in January 2006, because the pharmacologic management of bacterial (co)-infections, the development of protective devices, the improvement in intensive care techniques, the development of corticosteroids and active antibiotics, the antibody levels against various influenza strains in the global population and the communication channels to aware and teach populations on the health risks did dramatically evolve in the last 30 years, the drafting of pandemic forecasts and the prediction of global sanitary catastrophes (including the forecast of numbers of potential casualties) by mathematic models combining data obtained from the history of epidemiology with viral gene reassortments should be considered as a maximalist panic-triggering non-sense.



The approaches to protect populations

Active protection with vaccines

Influenza vaccines (viral antigens) contain whole viral preparations obtained from allantoic fluids harvested from infected egg cultures, inactivated by formalin or lactones, mixed with adjuvant and administered by parenteral route. Vaccines induce humoral responses (humoral= immunoglobulins or antibodies), which protects from sickness and death.

Even if arguments for the use of vaccines are advocated for poultry on the basis of economic and social consequences of influenza outbreaks, it was shown that chickens immunised against highly pathogenic viruses might carry and excrete virulent variants following challenge with live virus. Because the incomplete prevention of virus excretion and the immune pressure can generate variants, the vaccines produced with low and high pathogenic H5 andH7 viruses had been prohibited in the USA between the 80’s and 1995 (the distinction between vaccinated from infected animals could mask the presence of high pathogenic viruses and allow its transmission).

If the next influenza pandemic were to begin the inactivated vaccines would offer a rapid means for mass human protection, but the production of this vaccine (obtained by viral expansion in embryonated chicken eggs) is time consuming and dependent on a steady supply of biological material since at least six months may be necessary to organize sufficient fertilized eggs for manufacturing a vaccine. Additional problems to be solved while producing vaccines refer to the high pathogenicity of the H5 strains that cannot be easily grown in large quantities because they are lethal to chicken embryos.

To complicate this picture it has to be also understood that because vaccines may induce adverse reactions in a non negligible number of recipients, the product liability and the unavailability of reasonable cost liability insurances in industrialized countries discourage manufacturers of vaccines for what’s called universal use. It was indicated that no new vaccine approach seems to trigger the interest of manufacturers for universal use unless the health authorities implement new mechanisms that limit liability for human adverse effects.

Antivirals

The H5N1 strains responsible for what is called “bird flu” are susceptible in-vitro to Neuraminidase inhibitors and in this family, two pharmacological agents, Oseltamivir (150 mg, two daily for 5 day) or Zanamivir (two inhalations in the morning and in the night for five days for 12 years or older).

For the anti-influenza compounds a “furor curandis optimism” was perceived when the information regarding these agents was released, but the fact of the matter is that up to this date no controlled clinical trials did prove the real levels of efficacy of any of the antivirals in humans exposed to or clinically affected with the H5N1 influenza virus. The therapeutic conclusions were built on pharmacological amalgams, empirical approximations and “good wishes” from data obtained in-vitro and on animal models known to have a certain predictive value for the clinical efficacy of antivirals in humans. It has to be emphasized that the recommended anti-influenza compounds, whose potential benefits are massively diffused by the media (and for the first time in the history of antiviral pharmacology using brand names!) "may be" active in human settings against the virus responsible for bird flu and that these drugs “may be” useful in protecting health workers and other exposed individuals.


The passive therapeutic approach

The experiences of colleagues who had to face previous outbreaks of viral diseases should inspire the design of strategies for treatments against the bird flu. On this matter it has to be pointed out that Junin's virus (Arenavirus) was responsible for several years in Latin America (Argentina) for a hemorrhagic fatal fever with high levels of mortality. This virus infects humans by aerosols (like Influenzaviruses) but from a reservoir in rodents, and without treatment the mortality reaches levels equivalent to that reported for the bird flu Influenza H5N1.

In 1975 several investigators reported that animals treated with hyper immune sera in appropriate doses (passive immunotherapy) showed significantly lower mortality rates than those treated with sera from animals with no antibodies (the reduction in mortality did dramatically diminish from 100% to 25%). The hyper immune sera were obtained from survivor animals during the phase of convalescence (animals that were sick but able to survive thanks to their ability to synthesise high levels of neutralizing antibodies). It was then shown that the administration of hyper immune sera in the phase of incubation or in the acute phase did improve the prognosis. It was also shown that the antibodies present in the mothers’ milk from mice previously immunized were able to reduce the out spring morbidity and mortality of animals challenged.

Clinical trials carried out with passive antiviral therapeutics, that could be today considered an “old fashion” approach, showed in 1994 in humans with Junin’s haemorrhagic fever, that the mortality rates ranged between 15 % and 30% in non treated subjects but was reduced to levels close to zero in patients receiving hyper immune sera obtained and purified from convalescents. In addition to the benefits observed while treating Arenavirosis, a group of veterinarians in Ohio showed differences in mortality between the offspring of mothers previously hyper immunized and not hyper immunized (14% and 86% respectively) in piglets experimentally infected by the oro-nasal route with Coronaviruses responsible for gastroenteritis and fatal pneumonia (to this family of viruses belongs the agent responsible for SARS). In 2004 it was shown in Japan that the passive immunotherapeutic approach was able to protect animals from myocarditis caused by Influenza A virus, significantly reducing morbidity and increasing the survival rate. The passive immunotherapy with specific immunoglobulins also reduces significantly the morbidity and mortality in children with respiratory sincytial-virus (RSV) bronchiolitis. The different issues discussed above are strong enough to support the rational for studying the potential clinical benefits of the treatments with high-titres of specific immunoglobulin preparations on severe virus infections transmitted by aerosols.

To produce pharmaceuticals containing specific immunoglobulins the ex-vivo large scale-up approach can be applied, and here the purification of proteins produced in bioreactors after the incubation of genetic engineered cell-clones producing each one a specific neutralizing immunoglobulin directed to a N or H epitope has to envisaged (monoclonal cocktail), but the narrow activity of each monoclonal antibody and the risk of immune escape may limit their clinical interest. The approaches based on proteins extracted from cells need a comprehensive screening and may take several years before the population could be granted with the benefits of these “bio-tech” ex-vivo produced antibody mixtures.

An alternative (and inexpensive approach) can be drafted if in different countries we decide to cooperate and harmonise health policies. Here, inspired by the experience while struggling in the 70’s against Junin’s haemorrhagic fever, the survivors’ solidarity should be solicited in order to obtain their formal consent and to proceed to a scheduled collecting of sera (and/or lymphocytes). This appears as a simple and inexpensive alternative, but here, additional non-scientific “limitations” have to be verbalised. First, passive immunotherapy is not patentable and in the international context of the pharmaceutical and biotechnological industries the levels of profitability of plasmatic human derivatives do not tempt the ones deciding for new therapeutic developments. Second, the manufacturing of hyper immune sera and/or plasmatic proteins (obtained from convalescents) requires technologies that are in the public domain (added value is minimum) without royalties to be charged or paid. The preparation of these products requires minimal investments in equipment and most of the activities have to be carried-out in order to keep the records updated of the sick subjects who would be requested to donate plasma (and/or lymphocytes). The centres in the areas where the outbreaks are detected with new viral variants could then provide the plasma, the sera or circulating cells producing the antibodies.
Because flu peaks as a consequence of seasonal variations, the production of hyper immune sera could be forecast. The international and inter-seasonal cooperation should make possible to elaborate an efficient answer to the needs of countries at risk where new strains may be isolated. This issue could limit the expenses kept as provision for the stockpiling of expensive antivirals.


Conclusion

Presently, there is an urgent priority to verify the impact of the cross-reactivity induced by the available influenza-A vaccines on the morbidity and mortality that may provoke the new viral strains, because little is known if the antibodies directed to the viral preparations used previously for vaccination have a positive impact on the transmission or on the morbidity of the new variants.

Facing the risk of an eventual pandemic, all the approaches must be considered as complementary and non-excluding. However, a critical analysis suggests that clinical proofs for hypothetical efficacy of antivirals against bird flu in humans have to be placed at the same level that those found for preparations with specific hyper-immune gamma globulins. The extrapolation of results obtained for other pathogens in pneumology and in animal models with Influenzaviruses is a solid argument justifying the interest for developing affordable treatments based on preparations with high titres of specific antibodies (with safety requirements warranted). In the event of an avian flu epidemic in humans, the health workers should implement rapidly the best public health responses, and the rational measures (isolation, efficient mechanical barriers for biologic fluids, quarantine, availability of clinically active and affordable antivirals, production of vaccines and safe preparations containing high titres of human neutralizing antibodies) are part of the tools to bring future outbreaks under control.





Dr Pablo Goldschmidt
Laboratoire du Centre National d´Ophtalmologie des Quinze Vingts
28 rue de Charenton, Paris-France











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