Anti-Viral VaccinesMedicinal Chemistry

Donlene Webb

SMU

Viruses

A virus is a submicroscopic obligate parasitic particle that infects cells in biological organisms.

Viruses are non-living particles that can only replicate when an organism reproduces the virulent RNA or DNA.

Among other things, viruses do not move, metabolize, or decay on their own. Viruses are obligate intracellular parasites that lack the cellular machinery for self-reproduction.

Viruses infect eukaryotes and prokaryotes such as bacteria; bacteriophages. Typically viruses carry a small amount of genetic material, either in the form of

RNA or DNA, but not both, surrounded by some form of protective coat consisting of proteins, lipids, glycoproteins or a combination.

The viral genome codes for the proteins that constitute this protective coat, as well as for those proteins required for viral reproduction that are not provided by the host cell.

Viruses Viral nucleic acid can be DNA or RNA. It can be single or double stranded, circular or

linear, with most being linear. The nucleic acid is protected from physical, chemical and enzymatic damage by a protein

coat called a Capsid. Many viruses have a second envelope surrounding the Capsid on which there are spikes

with antigenic determinants. This outer surface of the virus is responsible for host cell recognition. Initially viral

proteins on the outer surface will attach to the hosts receptor molecules. A simplified viron is illustrated below.

Life Cycle

▪ Attachment, sometimes called absorption: The virus attaches to receptors on the host cell wall.

Injection: The nucleic acid of the virus moves through the plasma membrane and into the cytoplasm of the host cell. The capsid of a phage, a bacterial virus, remains on the outside. In contrast, many viruses that infect animal cells enter the host cell intact.

Transcription: Within minutes of phage entry into a host cell, a portion is transcribed into mRNA, which is then translated into proteins specific for the infecting phage.

Replication: The viral genome contains all the information necessary to produce new viruses. Once inside the host cell, the virus induces the host cell to synthesize the necessary components for its replication.

Assembly: The newly synthesized viral components are assembled into new viruses. Release: Assembled viruses are released from the cell and can now infect other cells, and

the process begins again.

Origin of Vaccines

Smallpox was the first disease people tried to prevent by purposely inoculating themselves with other types of infections. Inoculation is believed to have started in India or China before 200 BC. Physicians in China immunized patients by picking off pieces from drying pustules of a person suffering from a mild case of smallpox, grinding the scales to a powdery substance, and then inserting the powder into the person's nose in order for them to be immunized. In 1718, Lady Mary Wortley Montague reported that the Turks have a habit of deliberately inoculating themselves with fluid taken from mild cases of smallpox. Lady Montague inoculated her own children in this manner. In 1796, during the heyday of the smallpox virus in Europe, an English country doctor, Edward Jenner, observed that milkmaids would sometimes become infected with cowpox through their interactions with dairy cows' udders. Cowpox is a mild relative of the deadly smallpox virus. Building on the foundational practice of inoculation, Jenner took infectious fluid from the hand of milkmaid Sarah Nelmes. He inserted this fluid, by scratching or injection, into the arm of a healthy local eight year old boy, James Phipps. Phipps then showed symptoms of cowpox infection. Forty-eight days later, after Phipps had fully recovered from cowpox, Jenner injected some smallpox-infected matter into Phipps, but Phipps did not later show signs of smallpox infection

Timeline of Vaccines 18th century 1796 First vaccine for smallpox, first vaccine for any disease 19th century 1882 First vaccine for rabies 20th century 1932 First vaccine for yellow fever 1945 First vaccine for influenza 1952 First vaccine for polio 1954 First vaccine for Japanese encephalitis 1957 First vaccine for adenovirus-4 and 7 1962 First oral polio vaccine 1964 First vaccine for measles 1967 First vaccine for mumps 1970 First vaccine for rubella 1974 First vaccine for chicken pox 1977 First vaccine for pneumonia 1978 First vaccine for meningitis 1981 First vaccine for hepatitis B 1992 First vaccine for hepatitis A 1998 First vaccine for rotavirus

Vaccines The principle of vaccination is to induce a "primed" state in the vaccinated subject so that,

following exposure to a pathogen, a rapid secondary immune response is generated leading to the accelerated elimination of the organism and protection from clinical disease. Success depends on the generation of memory T and B cells and the presence in the serum of neutralizing antibody.

Attributes of a good vaccine 1.Ability to elicit the appropriate immune response for the particular pathogen:

Tuberculosis - cell mediated response most bacterial and viral infections - antibody

2. Long term protection    ideally life-long3. Safety    vaccine itself should not cause disease4. Stable    retain immunogenicity, despite adverse storage conditions prior to administration5. Inexpensive

Live Vaccines1. Live attenuated organisms Organisms whose virulence has been artificially reduced by in vitro culture under

adverse conditions, such as reduced temperature. This results in the selection of mutants which replicate poorly in the human host and are therefore of reduced virulence.  Replication of the vaccine strain in the host reproduces many of the features of wild type infection, without causing clinical disease.  Most successful viral vaccines belong to this group.

The immune response is usually good - when the virus replicates in the host cells, both antibody as well as cell mediated immune responses are generated and immunity is generally long lived.  Often, only a single dose is needed to induce long term immunity.

Schematic Diagram of Development of Attenuated Cell Strain

2. Heterologous vaccines Closely related organism of lesser virulence, which shares many antigens with the virulent organism. The vaccine strain replicates in the host and induces an immune response that cross reacts with antigens of the virulent organism. The most famous example of this type of vaccine is vaccinia virus:   Both cowpox virus and vaccinia virus are closely related to variola virus, the causitive agent of smallpox. Widespread use of vaccinia virus as a vaccine has lead to the world-wide eradication of smallpox.

Live Vaccines3. Live recombinant vaccines It is possible, using genetic engineering, to introduce a gene coding for an immunogenic protein from

one organism into the genome of another (such as vaccinia virus). The organism expressing a foreign gene is called a recombinant. Following injection into the subject, the recombinant organism will replicate and express sufficient amounts of the foreign protein to induce a specific immune response to the protein.

Attributes Good immune response Both Cell Mediated Immunity and antibody responses. Immunity is long lived Single dose Safety Danger of reversion to virulence, or Severe disease in immunocomprised Stability Organisms in the vaccine must remain viable in order to infect and replicate in the host Vaccine preparations are therefore very sensitive to adverse storage conditions Maintenance of the cold chain is very important. Expense Cheap to prepare

Vaccinia Virus The most unusual, and perhaps technologically the most useful, feature of poxviruses is

their ability to replicate in the infected cell's cytoplasm, and not nucleus. Infectious virions have a lipoprotein envelope surrounding a complex core of linear duplex DNA connected at each end by hairpin loops. Virus encoded enzymes, a multi-subunit DNA-dependent RNA polymerase, a transcription factor, capping and methylating enzymes, and a poly(A) polymerase are all contained within the core. Vaccinia is therefore well equipped to synthesize translatable mRNA.

Vaccinia VirusVaccinia undergoes homologous recombination during replication in infected cells. When used as an

expression vector, this innate ability to recombine is used to introduce foreign DNA coupled to a vaccinia promoter. The steps below outline the construction of the vaccinia expression vector.

(1) The gene (YFG) is flanked with vaccinia DNA sequences, especially the vaccinia promoters and multicloning sites for cleavage and ligation.

The following are often included: The promoters are necessary DNA sequences because the endogenous viral RNA polymerase binds

here to initiate transcription. The promoter also determines the direction of translation for the insert, and more importantly the ability to express proteins (depending on how tightly regulated the promoter is).

In addition, DNA sequences, such as the lacO/lacIq repressor system, that act in conjunction with promoters and also bind repressor molecules can regulate the induction of transcription. Hence, by adding or removing a particular substrate, expression of YFG can be turned on and off as necessary.

Stabilizing elements such as transcription terminators can also be incorporated downstream of the multicloning site. These anti-termination elements signal the RNA polymerase to release the DNA template and stop transcription, and prevent pausing, pre-mature termination, and overreading which adversely affect plasmid replication.

Finally, small open reading frames, known as ribosome binding sites, upstream of YFG, can be included to encourage binding and translation of the target sequence.

Vaccinia Virus(2) The product (usually a plasmid with an ori and a marker gene) is then inserted into a cell infected with

the whole virus. The whole virus must be used because it contains the necessary enzymes and factors within its core.

(3) Recombination during replication leads to insertion of YFG (i.e. the foreign DNA) into the viral progeny. The usual target of insertion is a nonessential region, so that virus retains its ability to replicate independently and the system can be maintained. The estimated incidence of successful insertion is approximately 0.1% (hey, I didn't say this was easy...). A major advantage of the vaccinia vector is that atleast 25,000 bp of DNA (a lot more than most vectors can handle) can be added to the vaccinia genome without requiring any deletions.

(4) Controlling when and how much of YFG is expressed is easy because the poxvirus promoter sequences control the rate and time of expression, and you can regulate which promoters are in the system. The highest yeilds of protein are generally generated with the late promoters.

(5) Virus plaques can finally be screened by DNA hybridization or for expression of your favorite protein.With the rapid discovery of new genes, especially from the Human Genome Project, comes the daunting

task of understanding how the products of these genes are synthesized, regulated, and used within cells. Vaccinia virus, as a vector for expression systems, is a powerful addition to the range of molecular methods available for such purposes. The use of Vaccinia allows temporal, as well as quantitative regulation of protein expression. More importantly, Vaccinia is large enough to accomodate several gene inserts while preserving the entire length of its DNA. Finally, as an infectious agent, it can target specific cells for insertion, and may thus be employed in gene and cancer therapy. Led by Vaccinia, the Poxviridae may no longer be considered the scourge of the world, but rather powerful tools for advancing research and therapeutic avenues.

Killed (inactivated) vaccines

When safe live vaccines are not available, either because attenuated strains have not been developed or else because reversion to wild type occurs too readily, it may be possible to use an inactivated  preparation of the virulent organism to immunize the host.

The organism is propagated in bulk, in vitro, and inactivated with either beta-propiolactone or formaldehyde. These vaccines are not infectious and are therefore relatively safe. However, they are usually of lower immunogenicity and multiple doses may be needed to induce immunity. In addition, they are usually expensive to prepare.

Subcellular fractions When protective immunity is known to be directed against only one or two proteins of an organism, it

may be possible to use a purified preparation of these proteins as a vaccine. The organism is grown in bulk and inactivated, and then the protein of interest is purified and concentrated from the culture suspension. These vaccines are safe and fewer local reactions occur at the injection site. However, the same disadvantages of poor immunogenicity and the need for multiple boosters applies.

Recombinant proteins Immunogenic proteins of virulent organisms may be synthesized artificially by introducing the gene

coding for the protein into an expression vector, such as E-coli or yeasts. The protein of interest can be extracted from lysates of the expression vector, then concentrated and purified for use as a vaccine. The only example of such a vaccine, in current use, is the hepatitis B vaccine.

Killed (inactivated) vaccinesAttributes Immune response

poor;  only antibody - no cell immediated immune response. response is short-lived and multiple doses are needed. may be enhanced by the incorporation of adjuvants into the vaccine preparation (see

below)

1. Safety Inactivated, therefore cannot replicate in the host and cause disease. Local reactions at the site of injection may occur. 2. Stability Efficacy of the vaccine does not rely on the viability of the organisms. These vaccines tend to be able to withstand more adverse storage conditions. 3. Expense Expensive to prepare

AdjuvantsCertain substances, when administered simultaneously with a specific antigen, will enhance

the immune response to that antigen. Such compounds are routinely included in inactivated or purified antigen vaccines.

Adjuvants in common use: 1. Aluminium salts First safe and effective compound to be used in human vaccines. It promotes a good antibody response, but poor cell mediated immunity. Form precipitate with antigen, making complex more antigenic2. Liposomes and Immunostimulating complexes (ISCOMS) 3. Complete Freunds adjuvant is an emulsion of Mycobacteria, oil and water Too toxic for man Induces a good cell mediated  immune response. 4. Incomplete Freund's adjuvant as above, but without Mycobacteria. 5. Muramyl di-peptide Derived from Mycobacterial cell wall. 6. Cytokines IL-2, IL-12 and Interferon-gamma. Possible modes of action: By trapping antigen in the tissues,  thus allowing maximal exposure to dendritic cells and

specific T and B lymphocytes. By activating antigen-presenting cells to secrete cytokines that enhance the recruitment of

antigen-specific T and B cells to the site of inoculation.

Subunit Vaccines

Immune response can be stimulated by one or a set of viral proteins. This was first demonstrated by hepatitis B and influenza vaccines

These can be a lot safer than attenuated or inactivated vaccines The subunits included are determined by identifying which proteins the

antibodies recognize. Subunits vaccines

Composed solely of purified protein can be delivered to body by means of a nonpathogenic virus, bacteria,

etc

DNA Vaccines

DNA vaccines are at present experimental, but hold promise for future therapy since they will evoke both humoral and cell-mediated immunity, without the dangers associated with live virus vaccines.

The gene for an antigenic determinant of a pathogenic organism is inserted into a plasmid.  This genetically engineered plasmid comprises the DNA vaccine which is then injected into the host.  Within the host cells, the foreign gene can be expressed (transcribed and translated) from the plasmid DNA, and if sufficient amounts of the foreign protein are produced, they will elicit an immune response.

in recent years a new type of vaccine, created from an infectious agent's DNA called DNA vaccination, has been developed. It works by insertion (and expression, triggering immune system recognition) into human or animal cells, of viral or bacterial DNA. These cells then develop immunity against an infectious agent, without the effects other parts of a weakened agent's DNA might have. As of 2006, DNA vaccination is still experimental, but shows some promising results.

Vaccines in General Use

Measles Live attenuated virus grown in chick embryo fibroblasts, first

introduced in the 1960's. Its extensive use has led to the virtual eradication of measles in the first world. In developed countries, the vaccine is administered to all children in the second year of life (at about 15 months). However, in developing countries, where measles is still widespread, children tend to become infected early (in the first year), which frequently results in severe disease. It is therefore important to administer the vaccine as early as possible (between six months and a year). If the vaccine is administered too early, however, there is a poor take rate due to the interference by maternal antibody. For this reason, when vaccine is administered before the age of one year, a booster dose is recommended at 15 months.

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Measles—United States, 1950-2002

Vaccines in General UseMumps Live attenuated virus developed in the 1960's.   In first world countries it is

administered together with measles and rubella at 15 months in the MMR vaccine. The current "Jeryl Lynn" strain of the mumps vaccine was developed by Dr.

Maurice Hillman from the mumps virus that infected his 5-year-old daughter (whose name was Jeryl Lynn). This vaccine, combined with rubella or both rubella and measles vaccines (MMR), has been widely used worldwide (300 million doses given) since it was approved by the FDA in 1967.

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Vaccines in General Use

Polio Two highly effective vaccines containing all 3 strains of poliovirus are in general use: The killed virus vaccine (Salk, 1954) is used mainly in Sweden, Finland, Holland

and Iceland. The live attenuated oral polio vaccine (Sabin, 1957) has been adopted in most

parts of the world;  its chief advantages being: low cost, the fact that it induces mucosal immunity and the possibility that, in poorly immunized communities, vaccine strains might replace circulating wild strains and improve herd immunity.  Against this is the risk of reversion to virulence (especially of types 2 and 3) and the fact that the vaccine is sensitive to storage under adverse conditions. - Orimune®

The inactivated Salk vaccine is recommended for children who are immunosuppressed.

3 types of live polio virus, magnesium chloride, amino acid, polysorbate 80, purified water, neomycin, sulphate, streptomycin, penicillin and monkey kidney cell cultures.

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Vaccines in General Use

Rubella Live attenuated virus. Rubella causes a mild febrile illness in children,

but if infection occurs during pregnancy, the fetus may develop severe congenital abnormalities. Two vaccination policies have been adopted in the first world. In the USA, the vaccine is administered to all children in their second year of life (in an attempt to eradicate infection), while in Britain, until recently, only post pubertal girls were vaccinated.  It was feared that if the prevalence of rubella in the community fell, then infection in the unimmunized might occur later - thus increasing the likelihood of infection occurring in the child-bearing years.  This programme has since been abandoned in Britain and immunization of all children is the current practice.

MMR — live measles virus, live mumps virus, live rubella virus, chick embryo, human foetal cells, neomycin, sorbitol, gelatine.

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Rubella—United States, 1966-2002

Vaccines in General UseRabies No safe attenuated strain of rabies virus has yet been developed for humans.

Vaccines in current use include: The neurotissue vaccine - here the virus is grown in the spinal cords of

rabbits, and then inactivated with beta-propiolactone. There is a high incidence of neurological complications following administration of this vaccine due to a hypersensitivity reaction to the myelin in the preparation and largely it has been replaced by

A human diploid cell culture-derived vaccine (also inactivated) which is much safer.

There are two situations where vaccine is given: a) Post-exposure prophylaxis, following the bite of a rabid animal:A course of 5-6 intramuscular injections, starting on the day of exposure. Hyperimmune rabies globulin may also administered on the day of exposure.

b) Pre-exposure prophylaxis is used for protection of those whose occupation puts them at risk of infection with rabies;  for example, vets, abbatoir and laboratory workers. This schedule is 2 doses one month apart ,and a booster dose one year later. (Further boosters every 2-3 years should be given if risk of exposure continues).

Rabies

Vaccines in General UseHepatitis B Two vaccines are in current use: a serum derived vaccine and a recombinant vaccine. Both

contain purified preparations of the hepatitis B surface protein. The serum derived vaccine is prepared from hepatitis B surface protein, purified from the

serum of hepatitis B carriers. This protein is synthesised in vast excess by infected hepatocytes and secreted into the blood of infected individuals. A vaccine trial performed on homosexual men in the USA has shown that, following three intra-muscular doses at 0, 1 and 6 months, the vaccine is at least 95% protective.

A second vaccine, produced by recombinant DNA technology, has since become available. Previously, vaccine administration was restricted to individuals who were at high risk of exposure to hepatitis B, namely: infants of hepatitis B carrier mothers, health care workers, homosexual men and intravenous drug abusers. However, hepatitis B has been targetted for eradication , and since 1995 the vaccine has been included in the universal childhood immunization schedule. Three doses are given;  at 6, 10, and 14 weeks of age. As with any killed viral vaccines, a booster will be required at some interval (not yet determined, but about 5 years) to provide protection in later life from hepatitis B infection as a venereal disease.

HEPATITIS B — Hepatitis B virus gene, aluminium hydroxide, mercury, formaldehyde. For the genetically engineered vaccine: aluminium hydrochloride, sodium chloride and mercury.

Hepatitis A A vaccine for hepatitis A has been developed from formalin-inactivated , cell culture-derived

virus. Two doses, administered one month apart, appear to induce high levels of neutralising antibodies.  The vaccine is recommended for travellers to third world countries, and indeed all adults who are not immune to hepatitis A.

Vaccines in General UseInfluenza Repeated infections with influenza virus are common due to rapid antigenic

variation of the viral envelope glycoproteins. Antibodies to the viral neuraminidase and haemagglutinin proteins protect the host from infection.   However, because of the rapid  antigenic variation, new vaccines, containing antigens derived from influenza strains currently circulating in the community, are produced every year.   Surveillance of influenza strains now allows the inclusion of appropriate antigens for each season.The vaccines consist of partially purified envelope proteins of inactivated current influenza A and B strains.

Individuals who are at risk of developing severe, life threatening disease if infected with influenza should receive vaccine.   People at risk include the elderly, immunocompromised individuals, and patients with cardiac disease. In these patients, protection from disease is only partial, but the severity of infection is reduced.

Varicella-Zoster virus A live attenuated strain of varicella zoster virus has been developed.  It is not

licensed in South Africa for general use, but is used in some oncology units to protect immuno-compromised children who have not been exposed to wild-type varicella zoster virus. Such patients may develop severe, life threatening infections if infected with the wild type virus.

Vaccines in General UseYellow Fever The 17D strain is a live attenuated vaccine developed in 1937. It is a

highly effective vaccine which is administered to residents in the tropics and travellers to endemic areas. A single dose induces protective immunity to travellers and booster doses, every 10 years, are recommended for residents in endemic areas.

VirusVaccine Brand

Name Type Route

Hepatitis A

Havrix Inactivated Intramuscular

VAQTA Inactivated Intramuscular

Hepatitis B

Recombivax Subunit Intramuscular

Engerix-B Subunit Intramuscular

Influenza

Fluzone Whole Inactivated Intramuscular

Fluzone, FlueShield Split-Virion Intramuscular

Fluvirin Subunit Intramuscular

Japanese Encephalitis JE-Vax Inactivated Subcutaneous

Measles Attenuvax Live Attenuated Subcutaneous

Mumps Mumpsvax Live Attenuated Subcutaneous

Polio

Orimune Inactivated Salk Subcutaneous

IPOL, PoliovaxLive Attenuated Sabin Oral

Rabies

HDCV Inactivated Intramuscular

RVA Inactivated Intramuscular

RabAvert Inactivated Intramuscular

Rotavirus RotaShield Live Attenuated Oral

Rubella Meruvax II Live Attenuated Subcutaneous

Varicella-Zoster Varivax Live Attenuated Intramuscular

Yellow Fever YF-Vax Live Attenuated Subcutaneous

Vaccine Controversy The public health benefits of vaccinations are exaggerated. Critics of

vaccination policy point out that the mortality rates of some illnesses were already dramatically reduced before vaccines were introduced, and claim that further reductions cannot immediately be attributed to vaccines.

Secondary and long-term effects on the immune system from introducing immunogens directly into the bloodstream are not fully understood.

The recommended vaccination schedule does not consider the cumulative effect of being exposed to multiple immunogens at the same time and at a young age.

At least some vaccine studies did not include such young children (e.g., 5 week old infants, 2 month old infants), yet vaccination schedules start with newborns. There can be a vast difference between the weight and all around development of a newborn baby versus a toddler, yet this is not accounted for.

claims diseases including leukemia, MS, sids, autism, and others were rare, and have increased coinciding with the increased use of vaccinations, and that this is due to vaccinations.

Opponents of current vaccination policy question if vaccinations actually create immunity against the targeted diseases because some people who have been vaccinated still contracted the illness.

Vaccine Controversy By not exposing children to common childhood illnesses, they may be

more susceptible to diseases at a point when their immune system is weakened.

Vaccinations contain chemical components that are known to be toxic, such as formaldehyde, aluminum in various compounds, acetone, glyceride, ethylene glycol, and neomycin when injected in large enough quantities.

As is true with any medication, adverse events to the vaccine (even when rare) may be worse than the disease itself.

There are a number of possible conflicts of interest that may affect the research design, findings, and opinions about vaccines, including financial interests of companies, the self-regulatory mechanism of medical doctors, and fear of the consequences should vaccines be found to be dangerous..

Some researchers hypothesize possible links between the increasing incidence of cancer and use of vaccines, suggesting links to the way vaccines may alter the cells in our bodies.

Latest Discoveries and News CHINA - The Chinese government has given the go-ahead to a Shanghai-based

pharmaceutical firm to begin clinical trials of Tamiflu, an anti-viral drug that is believed to be the best defense against bird flu in humans. The drug will be manufactured by Shanghai Sunve Pharmaceutical Co Ltd under a licensing arrangement with Swiss drug producer Roche. The study will try to find out if the Tamiflu produced by the Shanghai firm is as effective as that produced by Roche.

Smallpox

References AAPPublications.org - 'Thimerosal and the Occurrence of Autism: Negative Ecological

Evidence From Danish Population-Based Data' Pediatrics, Vol 112, No 3, September 2003 (Denmark study on autism rates)

BMJJournals.com - 'Comparative efficacy of three mumps vaccines', Matthias Schlegel, Joseph J. Osterwalder, Renato L. Galeazzi, Pietro J. Vernazza, British Medical Journal' Vol 319, No 352, August 7, 1999

BrianDeer.com - 'Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children' Andrew Wakefield, et al., The Lancet, Vol 351, No 9103, February 28, 1998

Read Congressional Research Service (CRS) Reports regarding vaccines JPandS.org (pdf) - 'Thimerosal in Childhood Vaccines, Neurodevelopment Disorders, and

Heart Disease in the United States', Mark Geier, M.D., Ph.D., and David Geier, B.A., Journal of American Physicians and Surgeons, Vol 8, No 1, Spring, 2003

Vaccine Information.org - 'Vaccine Information for the Public and Health Professionals' [11] Vaccine history from smithsonian institute http://www.coldcure.com/html/smallpox.html [article on timeline of vaccine history]

References http://www.sh.lsuhsc.edu/IntraGrad/micro/OLD-Micro289/2003/289%20PID%2003%20Va

ccines%20.DOC http://www.bio.davidson.edu/Courses/Molbio/MolStudents/01teparakh/Methods.htm http://web.uct.ac.za/depts/mmi/jmoodie/vacc2.html