Synthetic Replikins H1-H3-H5 Trans-Flu™ Vaccine Found Effective

Boston, September 30, 2009.

Figure. Virus Structure Predicted Outbreak and Course of H1N1 2009 Pandemic

Replikins, Ltd. published a warning on April 7, 2008 (1#18), one year before the current outbreak, that as determined by FluForecast® software, the concentration of a group of genomic peptides in the H1N1 influenza virus in humans, replikins (2), which was increasing since 2001, had reached the level of 7 per 100 amino acids, (Figure, red), a concentration which had occurred previously in the H1N1 pandemic of 1918. In the first three months of 2009, H1N1 outbreaks were reported in Mexico and California, then expanded globally into the present H1N1 pandemic. Since April 2009, FluForecast® software methods have provided advance information on the changing virus structure as it predicts the clinical course of the H1N1 pandemic (Figure).

All data published on PubMed was analyzed for replikin concentration in the virus genomes from 1918 to the present worldwide. The number of specimens from which sequence data for the hemagglutinin and polymerase genomic areas could be obtained for analysis from 2001 through 2008 was 855; in 2009 alone to date, 1,561.

The expectation of a summer interruption in the pandemic was contradicted by the prediction of FluForecast® data in May 2009 (1#28); and infections proceeded throughout the summer in the UK, China, the U.S.., and many other Northern Hemisphere countries, where they were not expected, as well as in the Southern Hemisphere where they were. As expected, the peak Replikin Count, between December 2008 and April 2009, was followed two months later in June in the U.S. by the peak of H1N1 pediatric deaths (3). Before the discovery of the replikins, to our knowledge, no virus structure had been reported which quantitatively correlated with or predicted virus outbreaks or clinical course.

Replikins were found not to be distributed evenly throughout the genome but to be concentrated in two areas: one associated with lethality (in the polymerase area) and one with infectivity (in the hemagglutinin area).

The Figure shows that for the H1N1 Infectivity Gene (red), the Mean Replikin Count increased from 4.3 (+/-2) in 2001 to 6.7 (+/-1.2) in 2008 (p<.001), when the warning was published, and increased another 43% to 10 by April 2009, when the clinical H1N1 outbreak was reported. In June 2009, WHO stated that the outbreak had spread globally sufficiently to be declared a pandemic. As of September 2009 the Infectivity Gene Count has remained elevated, decreasing only 3% in its mean since the high in April, 2009, thus giving no significant sign as yet of an abatement, as occurred in SARS when a sharp drop in the Replikin Count of the spike protein in the year of the outbreak, 2003, signalled the abrupt end of the clinical outbreak.

The Figure also shows that for the H1N1 Lethality Gene (black), the Mean Replikin Count between 2001 to 2008, despite some activity, did not increase significantly as did the Infectivity Gene. However, the Standard Deviation of the Mean (SD), which is indicative that some viruses in the population have high Replikin Counts and are engaging in high replication rates, increased 5-fold between 2001 and December 2008, then between December 2008 and April 2009 the SD increased 45-fold over that of 2001. It has gradually decreased by 50% from its high in April 2009 to that on September 21, 2009 (p<.001)*(Figure). However, the mean Count is still 20% elevated, and the standard deviation of the mean, is still 25 times greater than the 'resting' level of 2001. This indicates that there are still active individual viruses within the currently circulating H1N1 virus population which contain increased Replikin Counts in their Lethality Genes, suggesting deaths are still possible. The overall trend, however, since April 2009, as seen in the Figure is clearly towards a return to the lower 'resting' Lethality Replikin Count of 2, which predominated from 1980 to 2008, or less than 2 which predominated from 1934 to 1979; these low Counts were generally accompanied by low clinical H1N1 lethality from 1934 to 2008. The increase in the Count of the Replikins in April 2009, together with the current statistically significant decline in the Lethality Gene Count, which was followed by the drop in H1N1 influenza-associated pediatric mortality since June 2009 (3), gain in significance against the background of low H1N1 Counts and low H1N1 influenza mortality from 1934 to 2008. Although high H1N1 infectivity is predicted to persist, there is no indication from the present data that a mortality rate even as high as that in April 2009 is to be anticipated. However, with the persistent high infectivity, the risk of serious disease remains.

New H5N1 Cycle Signaled by H9N2 Replikin Count Increase; Possible H5N1 - H1N1 Combination

As in H1N1, the replikins in H5N1 were found not to be distributed evenly throughout the genome but to be concentrated in the same two areas as in H1N1, and associated with lethality and infectivity. Clinically, H1N1 has high infectivity and low lethality, the opposite of H5N1, which has low infectivity and high lethality. Between 2004 and 2008, the Replikin Count of the H5N1 Infectivity Gene in birds has remained at approximately 5, whereas the mean Count of the Lethality Gene has risen more than seven-fold, from 2+/-0.1 in 2004 to 15.1+/-6.5 in 2008, and to as high as 30.0 in the precursor of H5N1, H9N2, in chickens(1#24). The integrity and independence of infectivity and lethality genes is thus established by their opposite activities, in one host, humans, as seen in the Figure, and in two different hosts, humans vs. birds (the test of 'Double Discrimination').

A proof of principle that it is possible to quantify the lethality of a virus by counting the number of replikins per 100 amino acids in the virus genome is supported in the present results. This proof of principle was first achieved in a blind predictive study of the relative lethality of four strains of Taura Syndrome virus (1#10). The applicability of this principle then was realized when this virus was blocked by a Syndrome virus specific synthetic replikins vaccine, protecting shrimp 91% from lethality (1#17). In another example, an increase in the lethality of H5N1 in human cases in 2007-08 was predicted in advance by the strain-specific Replikin Count of the Lethality Gene of H5N1 (1#11). Similarly, by comparing the Replikin Counts of the Lethality Genes of H5N1 Genes in eight Asian countries in 2006, the geographic site which would be first and worst struck in 2007 was correctly predicted as Indonesia (1#11,12).

The independent function of the Replikin Lethality and Infectivity genes demonstrated by the H1N1 quantitative Replikin Count may explain the surprise expressed by some that the mortality rate has not increased, and according to the latest replikins data, may be expected to decrease further.

Because of the known ability of segments of the genomic sequences to transfer between influenza strains, the possibility that the high infectivity of H1N1 might be combined with the high lethality of H5N1 is of concern, and there is apparently no method available to predict the probability of this occurence.

Replikins TransFlu™ Vaccine

To prepare in advance for the possibility of an H1N1-H5N1 combination occurring, a synthetic replikins vaccine named Replikins TransFlu™ , because it is based on shared replikins structures in the common A Influenza strains, has been developed by Replikins, Ltd. The synthetic TransFlu™ Vaccine is based on replikins found to be structurally shared between H1N1, H5N1, H9N2, and H3N2.

Trans-Flu Vaccine testing has begun, and the vaccine is available for further testing. A recent test by Jackwood et al at the University of Georgia of replikins TransFlu™ vaccine was successful against H5N1 in chickens (see below); and tests against H1N1 and H5N2 are following. Consumption of virus excretions by neighboring chickens is a major mechanism by which influenza reservoirs are maintained both in Asia and North America; this constant transfer could not be blocked by previous massive vaccine efforts in China. In the recent peer-reviewed publication (4), synthetic Replikins TransFlu™ vaccine, produced in 7 days, was shown to be effective in decreasing infectivity and completely blocking excretion of LPAI H5N1 in chickens. The publication concludes: "Taken together, these data indicate that a replikin peptide vaccine specifically made against the H5N1 Black Duck/NC/674-964/06 and administered 3 times to the upper-respiratory tract, was capable of protecting chickens from infection and shedding of the homologous virus, which is extremely important because reduced virus shedding and transmission decreases the potential for H5 LPAI viruses to become HPAI viruses. The study is also important because it shows that the vaccine can be effectively mass delivered to the upper-respiratory tract."

In defending against rapidly emerging and changing infectious diseases, the possibility has thus been introduced that many or all vaccines will be produced timely in the future by organic chemical synthesis, with the goal of avoiding both the 'too little, too late' syndrome, evident in vaccine production for the current H1N1 pandemic, and the side effects of biological vaccines.

Notes

* Dates in the Figure are PubMed publication dates, which are from one to four months after the dates the specimens were actually collected. The time difference represents the time taken for sequence analysis, review and publication. 'Real-time' replikin analysis of the specimens could more accurately be realized if it is found possible in the future to shorten the time for sequence analysis and publication.
** In 1976, a localized H1N1 outbreak led to the administration of millions of doses of vaccine because of the fear that a second H1N1 pandemic was beginning (5). Analysis of the recorded H1N1 virus genomic sequences of 1976 showed that the Replikin Count of the Lethality Gene was only 1.9+/-0. If this information about the H1N1 virus structure had been known in 1976 might it have influenced the reaction to the outbreak?

1. Replikins Ltd. Website : Replikins report numbers (#1-30).
2. Bogoch,S. Bogoch,ES. Replikins: the Biochemistry of Rapid Replication. Monograph, Begell House, New York. 2005.
3. CDC FluView, Week 36 and 37, ending September 12th and 19th, 2009.
4. Jackwood, et al. Efficacy of a Replikin Peptide Vaccine Against Low Pathogenicity Avian Influenza H5 Virus. Avian Diseases, Publication Online: doi: 10.1637/8892-042509-ResNote.1; Hard copy Article In Press. July 2009.
5. Sencer, DJ, Miller,JD. Emerging Infectious Diseases. www.cdc.gov/eid. Vol 12, No. 1, January 2006.

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