Method of rapidly producing improved vaccines for animals   

Abstract: A method of quickly producing a vaccine for a biotype of pathogenic microorganism is described, where a nucleic acid molecule or fragment thereof is obtained from a biological sample from an animal exposed to the microorganism, a protective molecule is prepared based on the nucleic acid molecule of interest or fragment thereof, and administered to an animal which has been or is as risk of being exposed to the microorganism. A protective response to the biotype of the microorganism is obtained in the animal.

This application claims priority to previously filed and co-pending provisional application U.S. Ser. No. 61/407,297, filed Oct. 27, 2010; to previously filed and co-pending provisional application U.S. Ser. No. 61/418,433, filed Dec. 1, 2010 to previously filed and co-pending application U.S. Ser. No. 61/449,940 filed Mar. 7, 2011; to previously filed and co-pending application U.S. Ser. No. 61/484,255 filed May 10, 2011; to previously filed and co-pending application U.S. Ser. No. 61/508,172 filed Jul. 15, 2011; and to previously filed and co-pending application U.S. Ser. No. 61/525,332 filed Aug. 19, 2011, the contents of each of which are incorporated herein by reference in their entirety.

This invention was made with Government support under contract 2007-33610-18035 and 2009-33610-20299 awarded by and SBIR grant from the U.S. Department of Agriculture. The Government has certain rights in the invention.

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 4, 2011, is named 150003US.txt and is 103,997 bytes in size.

Animal vaccines are commonly produced by several routes. Commercially developed vaccines are produced for use in all animals, in many different locations. Such a vaccine would contain antigen and could be derived from a biotype of the pathogenic agent. Different isolates can be biotyped by a variety of typing techniques where a variant of the pathogenic agent is distinguishable by a particular characteristic over other members of the pathogenic species. These variants may differ, for example, by sequence variation of a DNA sequence, RNA sequence, pathogenic response, serological type or the like. See Sambrook et al, Molecular Cloning: A Laboratory Manual, Third editions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 2001. Another example of biotyping is glycan typing as is described at Harris et al., U.S. Pat. No. 7,622,254, incorporated herein by reference in its entirety. In the example of influenza virus, the viral strains are analyzed genetically and antigenically by the World Health Organization and the Center for Disease Control by screening numerous influenza viruses circulating in the human and animal populations. A vaccine strain is updated when there is an antigenic difference between the vaccine strain and newly emerged strain of two units in the hemagglutination-inhibition assay. Autogenous vaccines on the other hand are those produced for use at a particular location and group of animals. The pathogenic agent such as a virus is isolated and the whole virus used in producing a vaccine customized for the biotypes found at that location and which may be used at other locations where there may be exposure to such biotype. By way of example, an autogenous vaccine in a veterinary setting may be developed by isolating a virus at a farm to be used as a vaccine at the farm.

The present vaccine options for animals lack an ability to adapt quickly to new outbreaks of disease. Livestock animal diseases cost producers billions of dollars each year in both treatment and lost productivity. For certain diseases, such as those affecting aquatic invertebrates, there is no commercially available vaccine for the key pathogens (such as those affecting farmed shrimp). Porcine Reproductive Respiratory Virus (PRRSV) alone costs the swine industry an estimated $560 M annually. Neumann E J, Kleibenstein J, Johnson C, Mabry J W, Bush E J, Seitzinger A H, Green A L, Zimmerman J J (2005), Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States, Journal of the American Veterinary Medical Association 227: 385-392. Swine producers and veterinarians lack a broadly-effective vaccine for PRRS due to the lack of cross-protection commercial vaccines provide against heterologous strains of the pathogen. Influenza is another example of costly disease that affects humans, birds, swine and other animals. Veterinarians identified influenza as a swine disease during the influenza pandemic of 1918 when a connection was made between outbreaks in humans and swine that were closely related in time. According to the International Society for Infectious Diseases, the virus was initially isolated from pigs in 1930, with isolation from humans following in 1934. Influenza is grouped into three categories, based on the absence of serologic cross reactivity between their internal proteins: influenza A, B and C. Influenza A viruses are further classified into groups by antigenic differences of hemagglutinin and neuraminidase proteins. There are sixteen subtypes of HA and nine of NAs known, including H1, H2, H3, N1 and N2. The influenza viruses change frequently as a result of changes in the HA and NA amino acid sequence, allowing the virus to escape being neutralized by the immune response of the body. For example, to date the more prevalent subtypes circulating in human, poultry and swine populations in North America are H1 and H3. In 2008/2009 a new isolate was discovered which has resulted in another pandemic among humans, known as H1N1. This is why when vaccines are prepared from a particular strain it may not provide protection against an outbreak from a different strain, thus requiring vaccines to be prepared anew each year from predicted new or expected isolates.

Thus a vaccine specific for a specific biotypes of pathogens in a particular location such as on a farm is highly desirable. Companies exist which specialize in such autogenous vaccines. However, currently all autogenous vaccines are whole-organism preparations which must be inactivated (killed vaccines). In brief, a pathogen is isolated from an animal/farm, purified, grown in a laboratory, formulated, and returned to the farm for vaccination of animals. These traditional autogenous vaccines attempt to address the problem of strain variation; however they are not produced fast enough to have an immediate impact on production and economic losses caused by the disease and are not compatible with diagnostic tests for differentiating infected from vaccinated animals (DIVA).

Vaccine strains in USDA Center for Veterinary Biologics approved commercial vaccines can be ‘switched out’ in approximately 12 months. Rapp-Gabrielspm V J, Sornsen S, Nitzel G, et al. Updating swine influenza vaccines. AASV 39th Annual Meeting Proceedings 2008; 261:264. Vaccine strains in autogenous vaccines can be ‘switched out’ more quickly, possibly within 3-6 months; however, the cost of preparing vaccines with new and updated strains for limited orders may limit availability. Order size may increase with regional/adjacent autogenous networks but may be complicated to organize and implement. Henry S., Swine influenza virus—efforts to define and implement regional immunization. AASV Proceedings 2009; 475:478. VCPR vaccines may be authorized/produced by veterinarians/producers. ‘Switch out’ of vaccine strains can be done more rapidly than by either USDA CVB approved commercial or autogenous vaccines. (See 9 CFR 107.1 regarding Veterinary Client Patient relationship arrangements (VCPR) and 9 CFR 113.113 regarding autogenous vaccines; see also Ryan Vander Veen, Kurt Kamrud, Mark Mogler, et al. Rapid Development of an Efficacious Swine Vaccine for Novel H1N1. PLoS Currents Influenza 2009 Nov. 2RRN1123.) In sum, there are at least three major deficiencies in current vaccines. 1. Specificity. Commercial vaccines do not account for strain variation of pathogens that change quickly in the field. Strain variation and lack of adequate cross-protection lead to vaccine failure and thus production/economic losses. 2. Timeliness. It can take one year to change a commercial vaccine to account for strain variation. Current autogenous vaccines seek to address the problem of specificity, but can take months to prepare from the time a new strain is identified to the time the first animals are vaccinated. For example, traditional killed autogenous vaccines for PRRSV currently available can take up to six months to prepare. In this time frame a new strain of the pathogen can emerge, thus decreasing the value/effectiveness of the autogenous vaccine. A faster way to produce autogenous vaccines is thus needed. 3. Components. All current autogenous vaccines are whole-organism in that the entire pathogen is included in the vaccine. In most diseases this is unnecessary since the specific antigens needed for protection are already known. Also, since the whole virus is contained in the vaccine, they are not compatible with differential diagnostic tests.

Thus there is a need for improved vaccines for animals.

All references cited herein are incorporated herein by reference. Examples are provided by way of illustration and not intended to limit the scope of the invention.

SUMMARY

A method of producing a vaccine is provided which protects the animal from adverse effects of a pathogenic microorganism. A biological sample is obtained from an animal which has been exposed to a microorganism, a nucleic acid molecule of interest or fragment thereof of the microorganism is obtained from the sample and a protective molecule produced from the nucleic acid of interest. The protective molecule may be a nucleic acid molecule comprising the sequence or a fragment of the nucleic acid molecule of interest, may be a polypeptide or fragment produced by the nucleic acid molecule of interest, or may be an RNA molecule that is antisense to the nucleic acid molecule of interest or forms a dsRNA that corresponds to all or a portion of the nucleic acid molecule of interest. Vaccines so produced and a method of protecting an animal using the vaccine are also provided.

FIG. 1 is a schematic representation of the autogenous recombinant protein vaccine production process.

FIG. 2 is a schematic representation of the Backbone Biological Agent pVEK DNA plasmid vector.

FIG. 3 is a schematic representation of the pVEK Plasmid Vector with Autogenous Donor Gene (ADG) insert.

FIG. 4 is a schematic representation of the Autogenous Donor Gene (ADG)/GOI and backbone biological agent/RNA replicon construction.

FIG. 5 is a schematic of the VEE genome organization and replication strategy.

FIG. 6 is a schematic of the VEE replicon particle vaccine and packaging system.

FIG. 7 is a diagram of the IMNV genome transcription and translation products showing regions targeted for RNAi, predicted protein products are indicated by dark gray lines or gray shading, with target regions for dsRNA production indicated as thick black lines.

FIG. 8 is a map of the vector pERK-3/M/GP5.

FIG. 9 is a graph showing total gross lung score in different treatment groups. Treatment groups with different letters are significantly different (ANOVA, p<0.05).

FIG. 10 is a graph showing interstitial pneumonia scores in different treatment groups. Treatment groups with different letters are significantly different (ANOVA, p<0.05).

FIG. 11 is a graph showing a summary of lung lymphoid hyperplasia scores. Treatment groups with different letters are significantly different (ANOVA, p<0.05).

FIG. 12 is a graph showing a summary of heart pathology scores. Treatment groups with different letters are significantly different (ANOVA, p<0.05).

FIG. 13 is a graph showing mean IDEXX ELISA S/P titer per group. Groups with different letters are significantly different (ANOVA, p<0.01) within day post challenge. The bars of each group are in the order listed: strict negative, placebo, ARP, inactivated and MLV.

FIG. 14 is a graph showing the number of pigs out of ten per group with a FFN titer ≧4. Groups with different letters are significantly different (Chi-square, p<0.05) within day post challenge. The bars of each group are in the order listed: strict negative, placebo, ARP, inactivated and MLV.

FIG. 15 is a graph showing geometric mean FFN titer by group. Groups with different letters are significantly different (ANOVA, p<0.01) within day post challenge. The bars of each group are in the order listed: strict negative, placebo, ARP, inactivated and MLV.

FIG. 16 is a graph showing live virus titration at 7 DPC. Groups with different letters are significantly different (ANOVA, p<0.01).

FIG. 17 is a graph showing the number of pigs out of ten per group PRRSV positive serum via RT-PCR. Groups with different letters are significantly different (Chi-square, p<0.05) within day post challenge. The bars of each group are in the order listed: strict negative, placebo, ARP, inactivated and MLV.

FIG. 18 is a Western blot confirming recombinant HA expression. Lane 1 is the ladder; lane 2 the Vero lysate (negative control); lane 3, recombinant HA (28.5 μg/ml); Lane 4: Recombinant HA (1.14 μg/ml); Lane 5: Recombinant HA (0.57 μg/ml); Lane 6: Recombinant HA (0.38 μg/ml).

FIG. 19 is a graph showing a study measuring neutralizing antibodies against FMDV A24 at defined days post vaccination or challenge. The control is the first bar in each grouping, Dose A is the second bar and Dose B the third bar.

FIG. 20 is a graph showing serial dilution of clarified inoculum diluted in 2% saline. Sham inoculation received an equivalent dose of 2% saline. N=20 shrimp per treatment.

FIG. 21 is a graph showing results of Example 4, Experiment 1. Shrimp were injected with 2 μg of dsRNA construct and challenged with IMNV 48 hours following administration. N=3 groups of 20 shrimp per treatment.

FIG. 22 is a graph showing results of Example 4, Experiment 2. Shrimp were inoculated with a serial dilution of dsRNA and challenged 48 hours following administration. N=10 shrimp per treatment.

FIG. 23 is a graph showing results of Example 4, Experiment 2. Shrimp were injected with a serial dilution of dsRNA and challenged 10 days following administration. N=10 shrimp per treatment.

FIG. 24A is a graph showing results of Example 4, Experiment 3. Shrimp were injected with 0.02 μg of dsRNA#3, 5′ truncate of dsRNA3, or a 5′ truncate of dsRNA3 and challenged 10 days following administration. N=3 groups of 10 shrimp per treatment.

FIG. 24B is a graph showing results of Example 4, Experiment 3, where shrimp were injected as in FIG. 24A, but with further truncates of dsRNA#3.

FIG. 25 is a graph showing results of Example 4, Experiment 4 Shrimp were inoculated with replicons or dsRNA and challenged 3 days following administration. N=3 groups of 10 shrimp per treatment.

FIG. 26 is a graph showing results of Example 4, Experiment 5 Shrimp were inoculated with replicon and challenged 3 days following administration. N=3 groups of 10 shrimp per treatment.

FIG. 27 is a graph showing results of Example 4, Experiment 5 Shrimp were inoculated with replicon and challenged 10 days following administration. N=3 groups of 10 shrimp per treatment.

FIG. 28 is a graph showing results of Example 4, Experiment 8 WSSV survival following primary challenge.

FIG. 29: Survival following secondary WSSV challenge 21 days following primary challenge.

FIG. 30 is a graph showing survival following vaccination via injection or reverse gavage. Animals were challenged 14 days post vaccination.

FIG. 31 is a graph showing survivorship of animals following challenge that were administered dsRNA 2 days post challenge. X-axis is days post challenge. Y-axis is percent survival. dsRNA3 and eGFP dsRNA groups were treated with 5 μg dsRNA and the challenge control was treated with an equivalent volume sterile water.

FIG. 32 is a graph showing percent survivorship of shrimp following treatment with feed containing different solutions. X-axis is days post infection with WSSV and Y-axis is percent of animals surviving. n=30 animals with 3 replicates of 10/treatment

FIG. 33 is a graph showing shrimp survival post-vaccination with dsRNA of varying lengths (day 0) and post IMNV infection (day 10). dsRNA target position on the IMNV genome and length are indicated in the key.

The invention relates to an improved vaccine to protect animals against a pathogen which can be prepared very rapidly and which addresses the problem of providing protection to animals against a new or evolving biotypes. The vaccine is a new type of autogenous vaccine, that is, it is created from a nucleic acid molecule derived from an infectious agent present on a specific farm, flock, herd, pond or geographic region. It is not necessary to isolate the infectious agent in the laboratory to obtain the gene. It is prepared from the nucleic acid of microorganism(s) present in an animal or a group of animals which have been exposed to a biotype of a pathogenic microorganism. Such animals from which the nucleic acid molecule is obtained are those living in an environment in which one can expect are likely to have been exposed to the same pathogen biotype. By way of example, without limitation, where such animals are livestock animals, they may be found living in a ranch, feed yard, farm, flock, pond or region and with sufficient contact such that one skilled in the art would expect such animals to have come into contact or are likely to come into contact with the same pathogen. Upon preparation of the autogenous vaccine, these animals would then be vaccinated with the vaccine. As provided for with by the American Veterinary Medical Association (AVMA), adjacent or non-adjacent groups of animals considered to be at risk may also be vaccinated. See www.avma.org/issues/policy/autogenous_biologics.asp “Guidelines for Use of Autogenous Biologics” (Oversight: COBTA; EB approved-1993; reaffirmed November 1997; reaffirmed April 2001; revised March 2006, November 2009). When referring to an autogenous vaccine is meant to include this current definition of the AVMA in which animals considered to be at risk may be vaccinated. Also, an individual animal, (often times a dog, cat, horse, or the like) may be the sole animal for which the autogenous vaccine is prepared. The source of the microorganism nucleic acid molecule of interest is any convenient biological sample such as animal tissue, fluid or cell which are expected to have the nucleic acid of the microorganism present, whether blood, skin, organ tissue, body fluids or the like.

The term biotype refers to distinguishing a pathogenic agent by one or more characteristics over other members of the pathogenic species. The invention is particularly useful in providing a process to quickly produce a vaccine that is useful with different and/or new biotypes of a pathogen, and in an embodiment is especially useful where a biotype is found in a particular group of exposed animals or with potential for exposure to that biotype. Using current methods, a vaccine that is available may not be helpful against a different or newly evolving biotype. This invention provides a process where a vaccine that is useful with the new or different biotype is quickly developed. A biotype variant of a species can be distinguished by a variety of one or more characteristics, such as ribosomal RNA sequence variation, DNA polymorphisms, pathogenic response, response of the exposed animal to a specific vaccine, serological typing of toxin production or many other possible variations depending upon the pathogenic agent (see e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Edit., Cold Spring Harbor Laboratory, cold Spring Harbor, N.Y. 2001; DNA cloning A Practical Approach, Vol. I and II, Glove, D. M. edit. IRL Press Ltd., Oxford, 1985; Harlow and Lane, Antibodies a Laboratory Manual, Cold Spring Harbor Publications, N.Y. 1988). By way of example, without limitation, influenza can be biotyped by distinguishing it by subtype and cluster. The category is determined by differences of their internal proteins, and further by differences of the hemagglutinin and neuraminidase proteins. A hemagglutinin inhibition test can be used, in one example, where a sample of specified dilution is applied to red blood cells and the titer determined by the maximum dilution that produces agglutination. Antibodies to the virus prevent attachment to red blood cells and thus hemagglutination is inhibited when antibodies to influenza virus are present. Results are reported as the reciprocal of maximum dilution that provides visible agglutination. See e.g., Katz et al. (2009) Morbid. Mortal. Weekly Rep. 58 (19) 521-524. Another example of biotyping is glycan typing where genotypes are grouped based on their glycosylation patterns. Such a process is described at Harris et al., U.S. Pat. No. 7,622,254, incorporated herein by reference in its entirety (see especially, for example, table 7 columns 48 and 49). For example, the strains of PRRSV (Porcine Reproductive Respiratory Virus) are classified based on whether they are European or North American strains. In another aspect of typing the PRRSV strains, the first letter is either EU (European like) or NA (North American like) to designate the genotype cluster. EU refers to isotypes of PRRSV characterized by conserved glycans at position 46, 53, or both in GP5. As used herein, NA refers to isotypes of PRRSV characterized by conserved glycans at position 44, 51, or both in GP5. Each strain is given a number corresponding to the number of glycosylation sites located in the ectodomain of GP5 amino acid sequence shown in Table 7 of \'254, but excludes highly conserved glycans located at aa44 and 51 for NA strains and aa46 and 53 for EU strains. Thus, NA-0 refers to the ectodomain of GP5 of NA strain that has no glycan and EU-0 refers to the ectodomain of GP5 of an EU strain that has no glycan. For example, NA-1 refers to the ectodomain of GP5 of a North American strain that has 1 glycan located on the ectodomain of GP5 excluding highly conserved glycans located at aa44 and 51 for NA strains. Table 1 represents such a glycantyping of PRRSV.

TABLE 1 PRRSV Glycantypea Number of predicted glycansb, c NA-0  0d NA-1 1 NA-2 2 NA-3 3 NA-4 4 NA-n n EU-0 0 EU-1 1 EU-2 2 EU-3 3 EU-4 4 EU-n n aNA+ North American, EU = European bNumber of glycans located on the ectodomain of GP5 excluding highly conserved glycans located at aa44 and 51 for NA strains and aa46 and 53 for EU strains. When these glycans are absent they should be noted as follows: if an NA-1 strain lacks a glycan at aa44 it is described as NA-1 (Δ44). cAs the number of predicted glycans increases so does the resistance to inducing protective (neutralizing) antibodies and/or susceptibility to such antibodies. NA-0 and EU-0 are predicted to be the parent strains for all NA and EU strains respectively. Thus these viruses should be included in attempts to generate cross-reacting antibodies. After NA-0 and EU-0, glycantyping may be a predictor of heterology which is currently poorly defined for PRRSV.

Any biotyping method to distinguish a pathogen from another of the species may be used in the invention.

The inventors provide for a new approach to the generation of autogenous vaccines. In current processes used, the whole organism is isolated, then attenuated or killed, and the animal vaccinated with the prepared virus. By way of example, U.S. Pat. No. 4,692,412 to Livingston et al. describes a method for preparing an autogenous vaccine for neoplastic diseases by mixing a sterile blood sample containing Progenitor cryptocides with sterile distilled water, incubating the admixtures, killing or inactivating the Progenitor cryptocides in the admixtures, microfiltering the admixture to remove blood cells and diluting the filture to form the vaccine. Here, rather than use the whole organism (either live or inactivated) as the vaccine, one uses only one or more microorganism individual gene(s) of interest (GOI) also referred to as the nucleic acid molecule of interest (NOI) or fragments thereof, derived from the pathogen and/or the protein such gene(s) encode that makes up the autogenous vaccine. Surprisingly, it is possible to produce a vaccine using such nucleic acid molecules and to provide an effective vaccine which protects the animal. The gene of interest refers to a nucleic acid molecule which may or may not represent an entire gene and may be one from which an RNA interfering molecule is produced, or encodes a polypeptide or fragment thereof that produces a protective and/or immune response in the animal when administered to the animal. As one skilled in the art appreciates, the actual vaccine uses a protective molecule and may contain the gene of interest or fragment thereof, or may contain the polypeptide or fragment thereof producing the protective response, or may contain the interfering RNA or may contain a combination.

For many pathogens the protective molecule(s) needed to induce protection are known. The gene of interest of a pathogen is first amplified from a diagnostic sample originating from the farm. While one can isolate and purify the pathogen, it is not necessary with this method. Not only in such an embodiment does this eliminate an unnecessary step and speed the production process, it removes the need to have an isolated pathogen. The gene may be isolated or any protective portion of it isolated by using any available method such as PCR. The gene is then used to prepare the vaccine and ultimately used to vaccinate animals that have been or may be exposed to the pathogen. These vaccines when compared to currently available vaccines would be faster, biotype specific, and compatible with diagnostic tests. These attributes would allow vaccines to quickly enter the market. By way of example, there are even further advantages in the livestock industry such as the swine industry or farmed aquatic invertebrates or in any other situations in which there is a high level of consolidation.

Further advances in quick production of such vaccines is achieved in one embodiment by producing the antigen via Replicon subunit (referred to in some instances as autogenous protein) or Replicon Particle (referred to in some instances as RNA particle) technology. Prior to using the NOI as a protective molecule or to produce a protective molecule, it must be obtained from the location of the disease outbreak (such as an individual farm or geographic region). Once that genetic information is obtained in an embodiment the Replicon Subunit or Replicon Particle approach may be used to generate an autogenous vaccine. In sum, one approach is to generate an RNA subunit (RS) vaccine by introducing the replicon RNA that expresses the NOI into cells in culture. Once the replicon carrying the NOI has been introduced into cells each of the individual cells express the NOI. Alphavirus replicon vectors express heterologous proteins at very high levels (up to 20% of the total cell protein). The protective antigen, derived from the NOI, that is expressed in the cells is harvested by lysing them. The NOI/cell lysate then constitutes the autogenous vaccine. Another approach is to generate an RNA particle (RP) vaccine. RP are produced by introducing into cell in culture a replicon RNA that expresses the foreign gene and two helper RNAs, one that codes for the alphavirus capsid protein and the other that codes for the alphavirus glycoproteins (E2 and E1). These RNAs can be introduced into cells using a number of methods such as lipid transfection or electroporation. After the three RNAs have been introduced into cells the replicon RNA replicates itself in-cis and the helper RNAs in-trans. The helper RNAs produce the structural proteins which recognize the replicon RNA and package it into RP. RP expressing the foreign gene constitutes the autogenous vaccine.

The methods and variations of same used to produce such replicons are known to one skilled in the art. Illustrative methodology can be found at U.S. Pat. No. 6,156,558, incorporated herein by reference in its entirety, and also at U.S. Pat. Nos. 6,521,235; 6,531,135; and U.S. Pat. Nos. 7,442,381; 6,541,010; 7,045,335; and 5,792,462 all of which are incorporated herein by reference in their entirety.

Alphavirus vectors and alphavirus replicon particles are used in embodiments of the invention. The term “alphavirus” has its conventional meaning in the art, and includes the various species of alphaviruses which are members of the Togaviridae family. This includes alphaviruses such as Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus, South African Arbovirus No. 86, Semliki Forest virus, Middelburg virus, Chikungunya virus, O\'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The viral genome is a single-stranded, messenger-sense RNA, modified at the 5′-end with a methylated cap, and at the 3′-end with a variable-length poly (A) tract. Structural subunits containing a single viral protein, C, associate with the RNA genome in an icosahedral nucleocapsid. In the virion, the capsid is surrounded by a lipid envelope covered with a regular array of transmembranal protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2. See Pedersen et al., J. Virol. 14:40 (1974). The Sindbis and Semliki Forest viruses are considered the prototypical alphaviruses, and have been studied extensively. See Schlesinger The Togaviridae and Flaviviridae, Plenum Publishing Corp., New York (1986). The VEE virus has also been studied. See U.S. Pat. No. 5,185,440 to Davis et al.

As the above patents illustrate, preparation of replicon subunits by using alphavirus replicon vectors to obtain polypeptides and using alphavirus replicon particles to produce protective molecules are processes known to one skilled in the art. There are many modification to the process available, and any process using a replicon subunit or replicon particle methodology can be used with the invention. In a certain embodiment an alphavirus replicon RNA vector that expresses the gene of interest in a host cell and the expressed product is harvested. In another embodiment a replicon RNA comprising the gene of interest is introduced into a cell along with two helper RNAs coding for the alphavirus capsid protein and for the glycoproteins. The replicon RNA is packaged into a Replicon Particle. This Replicon Particle can be the protective molecule.

Thus the system in one embodiment provides for infectious, defective, alphavirus particles, wherein each particle comprises an alphavirus replicon RNA, and wherein the replicon RNA comprises an alphavirus packaging signal, one or more heterologous RNA sequence(s), and a sequence encoding at least one alphavirus structural protein, and wherein the replicon RNA furthermore lacks a sequence encoding at least one alphavirus structural protein; wherein the population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture. For example, in U.S. Pat. No. 6,531,135, incorporated herein by reference in its entirety is shown in an embodiment an RP system which uses a helper cell for expressing an infectious, replication defective, alphavirus particle in an alphavirus-permissive cell. The helper cell includes (a) a first helper RNA encoding (i) at least one alphavirus structural protein, and (ii) not encoding at least one alphavirus structural protein; and (b) a second helper RNA separate from the first helper RNA, the second helper RNA (i) not encoding at least one alphavirus structural protein encoded by the first helper RNA, and (ii) encoding at least one alphavirus structural protein not encoded by the first helper RNA, such that all of the alphavirus structural proteins assemble together into alphavirus particles in the cell. Preferably, the alphavirus packaging segment is deleted from at least the first helper RNA.

There are many variations that are available to one skilled in the art when preparing such replicons. For example, in another embodiment described in the patent, the helper cell also includes a replicon RNA, which encodes the alphavirus packaging segment and an inserted heterologous RNA. In the embodiment wherein the helper cell also includes a replicon RNA, the alphavirus packaging segment may be, and preferably is, deleted from both the first helper RNA and the second helper RNA. For example, in the embodiment wherein the helper cell includes a replicon RNA encoding the alphavirus packaging segment and an inserted heterologous RNA, the first helper RNA includes the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, and the second helper RNA includes the alphavirus capsid protein. The replicon RNA, first helper RNA, and second helper RNA in an embodiment are all on separate molecules and are cotransfected into the host cell.

In an alternative embodiment, the helper cell includes a replicon RNA encoding the alphavirus packaging segment, an inserted heterologous RNA, and the alphavirus capsid protein encoded by the second helper RNA, and the first helper RNA includes the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein. Thus, the replicon RNA and the first helper RNA are on separate molecules, and the replicon RNA and the second helper RNA are on a single molecule. The heterologous RNA comprises a foreign RNA.

The RNA encoding the structural proteins, i.e., the first helper RNA and the second helper RNA, may advantageously include one or more attenuating mutations. In an embodiment, at least one of the first helper RNA and the second helper RNA includes at least one attenuating mutation. The attenuating mutations provide the advantage that in the event of RNA recombination within the cell, the coming together of the structural and non-structural genes will produce a virus of decreased virulence.

As another aspect a method of making infectious, replication defective, alphavirus particles is provided. The method includes transfecting a helper cell as given above with a replication defective replicon RNA, producing the alphavirus particles in the transfected cell, and then collecting the alphavirus particles from the cell. The replicon RNA encodes the alphavirus packaging segment and a heterologous RNA. The transfected cell further includes the first helper RNA and second helper RNA as described above.

As another aspect, a set of RNAs is provided for expressing an infectious, replication defective alphavirus. The set of RNAs comprises, in combination, (a) a replicon RNA encoding a promoter sequence, an inserted heterologous RNA, wherein RNA encoding at least one structural protein of the alphavirus is deleted from the replicon RNA so that the replicon RNA is replication defective, and (b) a first helper RNA separate from the replicon RNA, wherein the first helper RNA encodes in trans, the structural protein which is deleted from the replicon RNA and which may or may not include a promoter sequence. In this embodiment, it is preferred that an RNA segment encoding at least one of the structural proteins is located on an RNA other than the first helper RNA. Thus, for example, the set of RNAs may include a replicon RNA including RNA which encodes the alphavirus packaging sequence, the inserted heterologous RNA, and the alphavirus capsid protein, but both the alphavirus E1 glycoprotein and alphavirus E2 glycoprotein are deleted therefrom; and a first helper RNA includes RNA encoding both the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein.

In another embodiment, the set of RNAs also includes a second helper RNA separate from the replicon RNA and the first helper RNA. In this embodiment, the second helper RNA encodes, in trans, at least one structural protein, which is different from the structural protein encoded by the replicon RNA and by the first helper RNA. Thus, for example, the set of RNAs may include a replicon RNA including RNA which encodes the alphavirus packaging sequence, and the inserted heterologous RNA; a first helper RNA including RNA which may encode a promoter sequence and an RNA encoding both the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein; and a second helper RNA including RNA which encodes the alphavirus capsid protein, with the replicon RNA, the first helper RNA, and the second helper RNA being in trans from each other, on separate molecules.

As another aspect, is provided a pharmaceutical formulation comprising infectious alphavirus particles as described above, in an effective immunogenic amount in a pharmaceutically acceptable carrier. See, for example, the \'135 patent at column 2, line 10-column 11 line 52 which includes examples 1-5.

The phrases “structural protein” or “alphavirus structural protein” as used herein refer to the encoded proteins which are required for production of particles that contain the replicon RNA, and include the capsid protein, E1 glycoprotein, and E2 glycoprotein. As described hereinabove, the structural proteins of the alphavirus are distributed among one or more helper RNAs (i.e., a first helper RNA and a second helper RNA). In addition, one or more structural proteins may be located on the same RNA molecule as the replicon RNA, provided that at least one structural protein is deleted from the replicon RNA such that the replicon and resulting alphavirus particle are replication defective. As used herein, the terms “deleted” or “deletion” mean either total deletion of the specified segment or the deletion of a sufficient portion of the specified segment to render the segment inoperative or nonfunctional, in accordance with standard usage. See, e.g., U.S. Pat. No. 4,650,764 to Temin et al. The term “replication defective” as used herein, means that the replicon RNA cannot produce particles in the host cell in the absence of the helper RNA. That is, no additional particles can be produced in the host cell. The replicon RNA is replication defective inasmuch as the replicon RNA does not include all of the alphavirus structural proteins required for production of particles because at least one of the required structural proteins has been deleted therefrom.

The helper cell for production of the infectious, replication defective, alphavirus particle comprises a set of RNAs, as described above. The set of RNAs principally include a first helper RNA and a second helper RNA. The first helper RNA includes RNA encoding at least one alphavirus structural protein but does not encode all alphavirus structural proteins. In other words, the first helper RNA does not encode at least one alphavirus structural protein; that is, at least one alphavirus structural protein gene has been deleted from the first helper RNA. In one embodiment, the first helper RNA includes RNA encoding the alphavirus E1 glycoprotein, with the alphavirus capsid protein and the alphavirus E2 glycoprotein being deleted from the first helper RNA. In another embodiment, the first helper RNA includes RNA encoding the alphavirus E2 glycoprotein, with the alphavirus capsid protein and the alphavirus E1 glycoprotein being deleted from the first helper RNA. In a third, preferred embodiment, the first helper RNA includes RNA encoding the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, with the alphavirus capsid protein being deleted from the first helper RNA. The second helper RNA includes RNA encoding the capsid protein which is different from the structural proteins encoded by the first helper RNA. In the embodiment wherein the first helper RNA includes RNA encoding only the alphavirus E1 glycoprotein, the second helper RNA may include RNA encoding one or both of the alphavirus capsid protein and the alphavirus E2 glycoprotein which are deleted from the first helper RNA. In the embodiment wherein, the first helper RNA includes RNA encoding only the alphavirus E2 glycoprotein, the second helper RNA may include RNA encoding one or both of the alphavirus capsid protein and the alphavirus E1 glycoprotein which are deleted from the first helper RNA. In the embodiment wherein the first helper RNA includes RNA encoding both the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, the second helper RNA may include RNA encoding the alphavirus capsid protein which is deleted from the first helper RNA.

In one embodiment, the packaging segment or “encapsidation sequence” is deleted from at least the first helper RNA. In a preferred embodiment, the packaging segment is deleted from both the first helper RNA and the second helper RNA.

In an embodiment wherein the packaging segment is deleted from both the first helper RNA and the second helper RNA, preferably the helper cell contains a replicon RNA in addition to the first helper RNA and the second helper RNA. The replicon RNA encodes the packaging segment and an inserted heterologous RNA. The inserted heterologous RNA may be RNA encoding a protein or a peptide. The inserted heterologous RNA may encode a protein or a peptide which is desirously expressed by the host, alphavirus-permissive cell, and includes the promoter and regulatory segments necessary for the expression of that protein or peptide in that cell.

For example, in one preferred embodiment of the present invention, the helper cell includes a set of RNAs which include (a) a replicon RNA including RNA encoding an alphavirus packaging sequence and an inserted heterologous RNA, (b) a first helper RNA including RNA encoding the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, and (c) a second helper RNA including RNA encoding the alphavirus capsid protein so that the alphavirus E1 glycoprotein, the alphavirus E2 glycoprotein and the capsid protein assemble together into alphavirus particles in the host cell.

In an alternate embodiment, the replicon RNA and the first helper RNA are on separate molecules, and the replicon RNA and the second helper RNA are on a single molecule together, such that a first molecule, i.e., the first helper RNA, including RNA encoding at least one but not all of the required alphavirus structural proteins, and a second molecule, i.e., the replicon RNA and second helper RNA, including RNA encoding the packaging segment, the inserted heterologous DNA and the capsid protein. Thus, the capsid protein is encoded by the second helper RNA, but the second helper RNA is located on the second-molecule together with the replicon RNA. For example, in one preferred embodiment of the present invention, the helper cell includes a set of RNAs including (a) a replicon RNA including RNA encoding an alphavirus packaging sequence, an inserted heterologous RNA, and an alphavirus capsid protein, and (b) a first helper RNA including RNA encoding the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein so that the alphavirus E1 glycoprotein, the alphavirus E2 glycoprotein and the capsid protein assemble together into alphavirus particles in the host cell.

In one embodiment of the present invention, the RNA encoding the alphavirus structural proteins, i.e., the capsid, E1 glycoprotein and E2 glycoprotein, contains at least one attenuating mutation. The phrases “attenuating mutation” and “attenuating amino acid,” as used herein, mean a nucleotide mutation or an amino acid coded for in view of such a mutation which result in a decreased probability of causing disease in its host (i.e., a loss of virulence), in accordance with standard terminology in the art, See, e.g., B. Davis, et al., Microbiology 132 (3d ed. 1980), whether the mutation be a substitution mutation or an in-frame deletion mutation. The phrase “attenuating mutation” excludes mutations which would be lethal to the virus. Thus, according to this embodiment, at least one of the first helper RNA and the second helper RNA includes at least one attenuating mutation. In a more preferred embodiment, at least one of the first helper RNA and the second helper RNA includes at least two, or multiple, attenuating mutations. The multiple attenuating mutations may be positioned in either the first helper RNA or in the second helper RNA, or they may be distributed randomly with one or more attenuating mutations being positioned in the first helper RNA and one or more attenuating mutations positioned in the second helper RNA. Appropriate attenuating mutations will be dependent upon the alphavirus used. For example, when the alphavirus is VEE, suitable attenuating mutations may be selected from the group consisting of codons at E2 amino acid position 76 which specify an attenuating amino acid, preferably lysine, arginine, or histidine as E2 amino acid 76; codons at E2 amino acid position 120 which specify an attenuating amino acid, preferably lysine as E2 amino acid 120; codons at E2 amino acid position 209 which specify an attenuating amino acid, preferably lysine, arginine, or histidine as E2 amino acid 209; codons at E1 amino acid 272 which specify an attenuating mutation, preferably threonine or serine as E1 amino acid 272; codons at E1 amino acid 81 which specify an attenuating mutation, preferably isoleucine or leucine as E1 amino acid 81; and codons at E1 amino acid 253 which specify an attenuating mutation, preferably serine or threoinine as E1 amino acid 253.

In an alternate embodiment, wherein the alphavirus is the South African Arbovirus No. 86 (S.A.AR86), suitable attenuating mutations may be selected from the group consisting of codons at nsP1 amino acid position 538 which specify an attenuating amino acid, preferably isoleucine as nsP1 amino acid 538; codons at E2 amino acid position 304 which specify an attenuating amino acid, preferably threonine as E2 amino acid 304; codons at E2 amino acid position 314 which specify an attenuating amino acid, preferably lysine as E2 amino acid 314; codons at E2 amino acid position 376 which specify an attenuating amino acid, preferably alanine as E2 amino acid 376; codons at E2 amino acid position 372 which specify an attenuating amino acid, preferably leucine as E2 amino acid 372; codons at nsP2 amino acid position 96 which specify an attenuating amino acid, preferably glycine as nsP2 amino acid 96; and codons at nsP2 amino acid position 372 which specify an attenuating amino acid, preferably valine as nsP2 amino acid 372. Suitable attenuating mutations useful in embodiments wherein other alphaviruses are employed are known to those skilled in the art. Attenuating mutations may be introduced into the RNA by performing site-directed mutagenesis on the cDNA which encodes the RNA, in accordance with known procedures. See, Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985). Alternatively, mutations may be introduced into the RNA by replacement of homologous restriction fragments in the cDNA which encodes for the RNA, in accordance with known procedures.

To develop the autogenous recombinant proteins, specific primers are used to amplify gene(s) derived from the disease agent via polymerase chain reaction (PCR). The PCR products are then cloned into the pVEK expression system plasmid vector, creating a recombinant plasmid. The pVEK plasmid vectors are transcribed to create RNA. Electroporation of the purified RNA into Vero cells leads to the production of the autogenous recombinant proteins. The proteins are harvested by lysis of the electroporated cells using a nonionic detergent. A schematic of the production process may be found in FIG. 1. Specific primers, derived from the disease agent, are used to create a cDNA Autogenous Donor Gene (ADG), sometimes referred to as a Gene of Interest (GOI). The ADG is then cloned into a vector, such as the pVEK plasmid vector shown here, creating a recombinant plasmid. The pVEK recombinant plasmid is then transcribed, creating RNA. The purified RNA is electroporated into Vero cells, leading to the production of autogenous recombinant proteins. The proteins are harvested by cell lysis. The vaccine itself consists of autogenous recombinant protein(s) derived from genes whose sequence(s) are obtained from diagnostic isolates. The proteins are mixed with an adjuvant.

A synthetic plasmid vector is used called pVEK. The pVEK plasmid vectors incorporate derivatives of the nonstructural genes from the attenuated Venezuelan equine encephalitis VEE virus vaccine, TC-83. The pVEK plasmid vectors have been modified for optimal donor gene expression (Kamrud, K. I., Custer, M., Dudek, J. M., Owens, G., Alterson, K. D., Lee, J. S., Groebner, J. L. & Smith, J. F. (2007). Alphavirus replicon approach to promoterless analysis of IRES elements. Virology. 360, 376-87. Epub 2006 Dec. 6). The plasmids are linearized and RNA is transcribed from the plasmid DNA with T7 Express enzyme in the presence of Cap analog (Promega, Madison, Wis.) and purified. The purified RNA is electroporated into Vero cells (derived from Master Cells) for translation into autogenous recombinant proteins. Neither the pVEK plasmid vector nor the transcribed RNA contain the structural, capsid, or glycoprotein genes for TC-83.

The nonstructural genes aid in the expression of the autogenous recombinant proteins by forming a complex which transcribes additional autogenous recombinant gene RNA. Nsp1 serves as the capping enzyme and is believed to play a major role in the binding and assembly of the complex. Nsp2 is an RNA binding protein that has NTPase activity and likely functions as a RNA helicase to unwind duplex RNA. Nsp2 also functions as a protease that is required for post-translational processing of the nonstructural polyproteins. Nsp3 is a phosphoprotein whose role has not been determined. Nsp4 has been identified as the RNA polymerase. Rayner, J. O., Dryga, S. A. & Kamrud, K. I. (2002). Alphavirus vectors and vaccination. Rev Med Virol 12, 279-96.

The pVEK vectors control protein expression at the level of translation by incorporating internal ribosome entry site (IRES) elements associated with each donor gene (Kamrud, K. I., Custer, M., Dudek, J. M., Owens, G., Alterson, K. D., Lee, J. S., Groebner, J. L. & Smith, J. F. (2007). Alphavirus replicon approach to promoterless analysis of IRES elements. Virology. 360, 376-87. Epub 2006 Dec. 6) as depicted in FIG. 2. The sequence differs from the TC-83 genomic sequence by four mutations and the absence of all TC-83 structural genes. In addition, a kanamycin resistance Open Reading Frame has been inserted into the plasmid backbone as a selective marker to amplify E. coli that contain the replicon plasmid DNA. A multiple cloning site has also been inserted in place of the structural protein genes. The resulting plasmid, pVEK, is replicated in bacteria using the COLE1 origin of replication and contains a 5′ untranslated region, TC-83 nonstructural protein sequences, a 26S promoter, a multiple cloning site and a 3′ untranslated region, all placed downstream of a T7 polymerase promoter for in vitro RNA transcription. FIG. 3 shows the pVEK plasmid vector with the autogenous donor gene insert. Typically, restriction enzymes AscI and PacI are used to specifically digest the ADG cDNA at appropriate sites outside of the ADG prior to ligation with the pVEK plasmid vector. FIG. 4 is a schematic representation of the ADG insertion into the pVEK plasmid vector. Because the electroporated RNA produce the autogenous recombinant protein in the cytoplasm without any DNA intermediate forms, the autogenous recombinant protein vaccine avoids the concerns of chromosomal integrations. As such no nuclear recombination events can occur.

In another embodiment, replicon particle (RP) vaccines are prepared. The RP vector has numerous advantages for vaccine development including accurate production of native proteins, tropism for lymphoid cells, lack of viral replication and transmission, induction of mucosal and systemic immunity, sequential immunization potential, and lack of preexisting immunity to VEE in animals although they clearly can respond to the virus immunologically. Dickerman R W, Baker G J, Ordonez J V, cherer W F (1973), Venezuelan Equine Encephalomyelitis Viremia and Antibody Responses of Pigs and Cattle, American Journal of Veterinary Research 34: 357-361 The replication strategy of VEE is similar to that of other alphaviruses. Strauss J, Strauss E (1994), The alphaviruses: gene expression, replication, and evolution, Microbiol Rev 58: 491-562

From positive-sense genomic RNA, four non-structural proteins (nsP1-nsP4) are translated and function to replicate a full-length negative-sense RNA. The negative-sense RNA serves as a template for replication of additional genomic RNA, and for synthesis of a subgenomic messenger RNA (26S mRNA), produced in 10-fold molar excess compared to genomic RNA, which directs the synthesis of the VEE structural proteins. The structural proteins are translated initially as a polyprotein that is co-translationally and post-translationally cleaved to release the capsid (C) protein and the two mature envelope glycoproteins (E1 and E2). Since VEE is a positive-sense RNA virus, full-length cDNA clones of VEE can be used to generate RNA transcripts that, when introduced into susceptible cells, will initiate a complete virus replication cycle and generate infectious virus. Davis N L, Willis L V, Smith J F, Johnston R E (1989), In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant, Virology 171: 189-204.

Using site-directed mutagenesis of the DNA plasmid, VEE viruses can be generated containing mutations in the envelope glycoproteins that result in attenuated phenotypes. When inoculated into animals, such attenuated variants of VEE do not cause illness or significant viremia but are able to induce protective immunity against subsequent virulent VEE challenge in mice, horses and primates. Davis N, Powell N, Greenwald G, Willis L, Johnson B, Smith J, Johnston R (1991), Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cDNA clone, Virology 183: 20-31 Grieder F, Davis N, Aronson J, Charles P, Sellon D, Suzuki K, Johnston R (1995), Specific restrictions in the progression of Venezuelan equine encephalitis virus-induced disease resulting from single amino acid changes in the glycoproteins, Virology 206: 994-1006

Similarly, foreign genes can be inserted in place of the VEE structural protein gene region in the cDNA plasmid, and an RNA transcript from such a plasmid, when introduced into cells, will replicate and express the heterologous genes as shown schematically in FIGS. 5-6. This self-amplifying replicon RNA will direct the synthesis of large amounts of the foreign gene product within the cell, typically reaching levels of 15-20% of total cell protein. Pushko P, Parker M, Ludwig G V, Davis N, Johnston R E, Smith J F (1997), Replicon-Helper Systems from Attenuated Venezuelan Equine Encephalitis Virus: Expression of Heterologous Genes in Vitro and Immunization against Heterologous Pathogens in Vivo, Journal of Virology 239: 389-401 Because the replicon RNA does not contain the structural genes for VEE, it is a single-cycle, propagation-defective RNA and replicates only within the cell into which it is introduced. The replicon RNA can be packaged into RP by supplying the structural protein genes of VEE in trans (FIG. 5). Replicon RNA is packaged into RP when cells are co-transfected with replicon RNA and two separate helper RNAs, which together encode the full complement of VEE structural proteins. Pushko, supra. Importantly, only the replicon RNA is packaged into VRP, as the helper RNAs lack the packaging sequence required for encapsidation. Thus, the RP are propagation-defective, in that they can infect target cells in culture or in vivo, can express the foreign gene to high levels, but they lack critical portions of the VEE genome (i.e., the VEE structural protein genes) necessary to produce virus particles which could spread to other cells. The “split helper” system greatly reduces the chance of an intact genome being regenerated by RNA-RNA recombination and, the possibility of functional recombination with helper RNAs was further reduced by removal of the 26S promoter from helper RNAs altogether (Kamrud et al 2010 Development and Characterization of Promoterless Helper RNAs for Production of Alphavirus Replicon Particles. Journal of General Virology. 91:pp. 1723-1727). As an independent and additional layer of safety, attenuating mutations have been incorporated in the glycoprotein helper. (Pushko et al 1997) Journal of Virology 239:389-401. FIG. 6 shows the VEE replicon particle vaccine and packaging system process. Expression of the nucleic acid molecule of interest can be varied up or down by introducing spacer elements upstream of the IRES/NOI cassette but downstream of the 26S promoter. (Kamrud et al. 2007, “Alphavirus Replicon Approach to Promoterless Analysis of IRES Elements”, Virology 360(2), pp. 376-38′7) Also, where the gene of interest produces a potentially toxic protein, introducing a phosphoramidite morpholino oligomers at the same time the replicon and helper RNAs are electroporated into cells shuts down expression. The PMO blocks translation of the gene of interest during packaging of RP.

The vaccine of the present invention would be ideal for the following reasons: 1) The new vaccines do not contain live virus. Current modified live virus (MLV) vaccines could not be used in an eradication effort due to their ability to spread, revert to virulence, or recombine with field strains. Practices (such as “serum therapy” for PRRSV in pigs) in which live virus on a farm is deliberately spread and used to infect naïve animals will also not be part of a successful disease control and eradication program. 2) The new vaccines can differentiate infected from vaccinated animals. Current MLV and killed vaccines use the whole virus. Here the entire infective agent is not included. As an illustration, only a portion of the PRRSV is included in the RP vaccine allowing differentiation based on serology. For example, because the nucleocapsid is not part of the ARP vaccine the current IDEXX ELISA could be used to detect pigs/herds that were infected as opposed to just vaccinated. 3) The new vaccines are autogenous and can be produced quickly. Autogenous vaccines are desirable for animal disease treatment due to strain/biotype variation and lack of cross-protection. It is shown here that ARP vaccines can be produced faster (1 month and even less) than traditional autogenous vaccines (3 months) allowing for a quicker response.

In a yet further embodiment, one can optionally first determine if preparation of a new autogenous vaccine as described is more advisable by determining the antigenic drift of the pathogen. In such an embodiment, one obtains a sample comprising the microorganism as described above, and may optionally determine what biotype it is. Using influenza as an example, the subtype and cluster may be determined. In general, influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope lined by a matrix protein. The genome of influenza viruses is composed of eight segments of linear (−) strand ribonucleic acid (RNA), encoding the immunogenic hemagglutinin (HA) and neuraminidase (NA) proteins, and six internal core polypeptides: the nucleocapsid nucleoprotein (NP); matrix proteins (M); non-structural proteins (NS); and three RNA polymerase (PA, PB1, PB2) proteins. During replication, the genomic viral RNA is transcribed into (+) strand messenger RNA and (−) strand genomic cRNA in the nucleus of the host cell. Each of the eight genomic segments is packaged into ribonucleoprotein complexes that contain, in addition to the RNA, NP and a polymerase complex (PB1, PB2, and PA). As noted, influenza is grouped into three categories, based on the absence of serologic cross reactivity between their internal proteins: influenza A, B and C. Influenza A viruses are further classified into groups by antigenic differences of hemagglutinin and neuraminidase proteins. Examples of subtypes and further classification into clusters are shown in Table 2 below. The hemagglutinin antigens of influenza viruses change frequently in antigenic specificity as a result of changes in the HA and NA amino acid sequence.

TABLE 2 Subtype

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