15.4W: Vaccines

Active Immunization or Vaccination

The terms vaccination and vaccine derive from the work of Edward Jenner who, over 200 years ago, showed that inoculating people with material from skin lesions caused by cowpox (L. vaccinus, of cows) protected them from the highly contagious and frequently fatal disease smallpox. Since Jenner's time, the term has been retained for any preparation of dead or weakened pathogens, or their products, that when introduced into the body, stimulates the production of protective antibodies or T cells without causing the disease. In molecular terms, the goal is to introduce harmless antigen(s) with epitopes that are also found on the pathogen.

Vaccination is also called active immunization because the immune system is stimulated to develop its own immunity against the pathogen. Passive immunity, in contrast, results from the injection of antibodies formed by another animal (e.g., horse, human) which provide immediate, but temporary, protection for the recipient.

Kinds of Vaccines

Killed whole organisms

In this relatively crude approach, the vaccine is made from the entire organism, killed to make it harmless. The typhoid and cholera vaccines are examples.

Attenuated organisms

Here, the organism has been cultured so as to reduce its pathogenicity, but still retain some of the antigens of the virulent form. The Bacillus Calmette-Guérin (BCG) is a weakened version of the bacterium that causes tuberculosis in cows. BCG is used as a vaccine against tuberculosis in many European countries but is rarely used in the U.S.

Toxoids

In some diseases, diphtheria and tetanus are notorious examples, it is not the growth of the bacterium that is dangerous, but the protein toxin that is liberated by it. Treating the toxin with, for example, formaldehyde, denatures the protein so that it is no longer dangerous, but retains some epitopes on the molecule that will elicit protective antibodies.

Surface molecules

Antibodies are most likely to be protective if they bind to the surface of the invading pathogen triggering its destruction. Several vaccines employ purified surface molecules:

• Influenza vaccine contains purified hemagglutinins from the viruses currently in circulation around the world.
• The gene encoding a protein expressed on the surface of the hepatitis B virus, called hepatitis B surface antigen or HBsAg, can now be expressed in E. coli cells and provides the material for an effective vaccine. Hepatitis B infection is strongly associated with the development of liver cancer. Here then is a vaccine against a cancer.
• The genes encoding the capsid proteins of 9 strains of human papilloma virus (HPV) can be expressed in yeast and the resulting recombinant proteins are incorporated in a vaccine (Gardasil 9®). Because infection with some of these strains of HPV can lead to cervical cancer, here is another vaccine against cancer.
• Some 80 different strains of Streptococcus pneumoniae cause pneumonia in humans. They differ in the chemistry of the polysaccharide capsule that surrounds them and makes it difficult for phagocytes to engulf them by endocytosis. One current vaccine consists of tiny amounts of the purified capsular polysaccharides of the 23 most common and/or dangerous strains.

Inactivated virus

Like killed bacterial vaccines, these vaccines contain whole virus particles that have been treated (again, often with formaldehyde) so that they cannot infect the host's cells but still retain some unaltered epitopes. The Salk vaccine for polio (IPV) is an example.

Attenuated virus

In these vaccines, the virus can still infect but has been so weakened that it is no longer dangerous. The measles, mumps, and rubella ("German measles") vaccines are examples. The Sabin oral polio vaccine (OPV) is another example. It has advantages over the Salk vaccine in that

• it is given by mouth rather than by injection;
• the viruses it contains can spread to the other members of the vaccinee's family thus immunizing them as well.

It has the disadvantage that on rare occasions one of the strains in the vaccine regains full virulence and causes the disease. For this reason, the Salk vaccine has once again become the preferred vaccine worldwide.

Example: A new method of attenuation

The various attenuated-virus vaccines in current use were developed by rather hit-or-miss methods. However, scientists have been working on a technology exploiting the phenomenon of codon bias - that may make possible the rational development of safer vaccines.

One group, at Stony Brook University (see J.R. Coleman et al., Science, 27 June 2008), has engineered polio virus with hundreds of mutations in the genes encoding its capsid protein. However, every one of these is a "silent" mutation; that is, it simply changes the codon for the amino acid to a different codon for the same amino acid. When they created polio viruses in which pairs of new codons were ones that the wild polio virus avoids using (because its human host does), they found that the new viruses were far less infectious that the original. But note, that this procedure did not introduce any change in the amino acid sequence of the capsid protein. So one would expect that all the epitopes recognized by the immune system would be unchanged. And, indeed, they went on to show that mice immunized with the synthetic virus were protected from disease caused by the wild virus.

As mentioned above, one of problems associated with the attenuated live virus polio vaccine (Sabin) is the rare back mutation to full virulence. Such back mutation in these engineered viruses would be extremely unlikely considering the hundreds of silent mutations that would have to be reversed.

Summary Table: Here is a table describing some of the most widely-used vaccines used in humans.

Disease Agent	Preparation	Notes
Diphtheria	Toxoid	Often given in a single preparation DTaP (Daptacel®) for infants and young children; Tdap for teenagers and adults (the lower case letters signify the smaller amounts of the diphtheria and pertussis antigens in Tdap).
Tetanus	Toxoid
Pertussis	Killed bacteria ("P") or their purified components (acellular pertussis = "aP")
Polio	Inactivated virus	Inactivated polio vaccine: IPV (Salk)
	Attenuated virus	Oral polio vaccine; OPV (Sabin) Both vaccines trivalent (types 1, 2, and 3)
Hepatitis A	Inactivated virus	HAVRIX® and VAQTA®; also available in single shot with HBsAg (Twinrix®)
Hepatitis B	Protein (HBsAg) from the surface of the virus	Made by recombinant DNA technology
Rotavirus	Attenuated virus (Rotarix®) or 5 strains of the virus (RotaTeq®)	to prevent this serious diarrheal disease in infants
Human Papilloma Virus (HPV)	Protein from the capsid of 9 strains of the virus	Gardasil 9®; made by recombinant DNA technology
Diphtheria, tetanus, pertussis, polio, and hepatitis B	Uses acellular pertussis and IPV (Salk)	Pediarix®; combination vaccine given in 3 doses to infants
Diphtheria, tetanus, pertussis, polio, and Hemophilus influenzae type b (Hib)	Uses acellular pertussis and IPV (Salk)	Pentacel®; combination vaccine given in 4 doses to infants
Measles	Attenuated virus	Often given as a mixture (MMR) Do not increase the risk of autism. (Nor do any vaccines containing thimerosal as a preservative.)
Mumps	Attenuated virus
Rubella	Attenuated virus
Chickenpox (Varicella)	Attenuated varicella-zoster virus (VZV)	Also available combined with MMR ("MMRV" or ProQuad®)
Cholera	Killed bacteria	Three oral vaccines available
Influenza	Hemagglutinins	Contains hemagglutinins from the type A and type B viruses recently in circulation
	Attenuated virus	FluMist® — contains weakened viruses of the type B and two type A strains recently in circulation
Pneumococcal infections	Capsular polysaccharides	A mixture of the capsular polysaccharides of 23 common types. Works poorly in infants.
	13 capsular polysaccharides conjugated to protein ("PCV13")	Mobilizes helper T cells; works well in infants.
Meningococcal disease	4 polysaccharides conjugated to protein	To prevent outbreaks among new groups of young adults, e.g., college freshmen, military recruits
Hemophilus influenzae, type b (Hib)	Capsular polysaccharide conjugated to protein	Prevents meningitis in children
Rabies	Inactivated virus	Vaccine prepared from human diploid cell cultures (HDCV) or chick embryo cells (PCECV)
Smallpox	Vaccinia virus	Despite the global eradication of smallpox, is used to protect against a possible bioterrorist attack
Anthrax	Extract of attenuated bacteria	Primarily for veterinarians and military personnel
Typhoid	Three types are available: 1. killed bacteria 2. live, attenuated bacteria (oral) 3. polysaccharide conjugated to protein
Yellow fever	Attenuated virus
Tuberculosis	Attenuated bacteria (BCG)	Rarely used in the U.S.

Some of the Triumphs of Vaccination

The greatest triumph is the eradication of smallpox from the planet, with no naturally-occurring cases having been found since 1977. "Naturally-occurring" because one case (fatal) occurred later following the accidental release of the virus in a laboratory. As far as the public knows, smallpox virus now exists only in laboratories in the U.S. and Russia. There is currently a vigorous debate as to whether these should be destroyed. If smallpox ever should get back out into the environment, the results could be devastating because smallpox vaccination is no longer given and so the population fully susceptible to the disease grows year by year.

A program to try to eliminate polio from the world is now underway. Except for cases caused by OPV, the disease has now been eliminated from the Western hemisphere. Outbreaks of polio still occur in Africa, the Indian subcontinent, and parts of the Near East. Table 1 compares the number of cases of illness in the U.S. in a representative year (either before a vaccine was available or before it came into widespread use) with the number of cases reported in 1994.

Table 1

Disease	Total cases	Year	Cases in 1994	% Change
Diphtheria	206,939	1921	2	-99.9%
Measles	894,134	1941	963	-99.9%
Mumps	152,209	1968	1537	-99.9%
Pertussis	265,269	1934	4617	-99.9%
Poliomyelitis*	21,269	1952	0	-100%
Rubella	57,686	1969	227	-99.9%
Tetanus	1,560	1923	51	-99.9%

*Since 1979, an average of 8 cases of poliomyelitis have occurred in the U.S. each year that are acquired from the vaccine (OPV, the Sabin vaccine) itself. For this reason, the "killed" virus vaccine (IPV, the Salk vaccine) is being reintroduced. As of June 17, 1999, it is recommended that in the future all children receive 4 doses of the Salk vaccine and — except in special circumstances — none of the Sabin vaccine.

Problems of Vaccine Development

With so many triumphs, why haven't vaccines eliminated other common diseases such as malaria and HIV-1 infection?

One problem is that experimental vaccines often elicit an immune response that does not actually protect against the disease. Most vaccines preferentially induce the formation of antibodies rather than cell-mediated immunity. This is fine for those diseases caused by

• toxins (diphtheria, tetanus)
• extracellular bacteria (pneumococci)
• even viruses that must pass through the blood to reach the tissues where they do their damage (polio, rabies)

But viruses are intracellular parasites, out of the reach of antibodies while they reside within their target cells. They must be attacked by the cell-mediated branch of the immune system, such as by cytotoxic T lymphocytes (CTLs). Most vaccines do a poor job of eliciting cell-mediated immunity (CMI).

Example:

Much of the early and so far unsuccessful work on anti-HIV-1 vaccines has focused on the antibody response of the test animal. Antibodies may have a role in preventing infection or minimizing its spread, but cell-mediated responses will probably turn out to be far more important. Certainly there are thousands of patients dying of AIDS despite their high levels of anti-HIV-1 antibodies. The most widespread test for HIV-1 infection does not detect the presence of the virus but the presence of antibodies against the virus.

DNA Vaccines

With DNA vaccines, the subject is not injected with the antigen but with DNA encoding the antigen.

The DNA is incorporated in a plasmid containing

• DNA sequences encoding one or more protein antigens or, often, simply epitopes of the complete antigen(s);
• DNA sequences incorporating a promoter that will enable the DNA to be efficiently transcribed in the human cells.
• Sometimes DNA sequences encoding costimulatory molecules and sequences that target the expressed protein to specific intracellular locations (e.g., endoplasmic reticulum) are included as well.

The DNA vaccine can be injected into a muscle just as conventional vaccines are. In contrast to conventional vaccines, DNA vaccines elicit cell-mediated as well as antibody- mediated immune responses.

The cell-mediated response

• The plasmid is taken up by an antigen-presenting cell (APC) like a dendritic cell.
• The gene(s) encoding the various components are transcribed and translated.
• The protein products are degraded into peptides.
• These are exposed at the cell surface nestled in class I histocompatibility molecules where
• they serve as a powerful stimulant for the development of cell-mediated immunity.

The antibody-mediated response

• If the plasmid is taken up by other cells (e.g. muscle cells), the proteins synthesized are released and can be engulfed by antigen-presenting cells (including B cells).
• In this case, the proteins are degraded in the class II pathway and presented to helper T cells.
• These secrete lymphokines that aid B cells to produce antibodies.

So far, most of the work on DNA vaccines has been done in mice where they have proved able to protect them against tuberculosis, SARS, smallpox, and other intracellular pathogens. In addition, more than a dozen different DNA vaccines against HIV-1 the cause of AIDS are in clinical trials.