INFECTIOUS DISEASE

VIROLOGY -  CHAPTER  EIGHT  

VACCINES: PAST SUCCESSES AND FUTURE PROSPECTS

FROM SMALLPOX TO COVID-19

Dr Richard Hunt
Professor
University of South Carolina School of Medicine
Columbia
South Carolina

En Español

See also:
APPENDIX
Reduction in the incidence of certain diseases after the introduction of vaccination

 

Polio Virus (From the Hogle Lab at Harvard, URL unknown)

Many of the images in the smallpox part of this file come from Fenner, Henderson, Arita et al. Smallpox and its Eradication. 1988 Geneva, World Health Organization and were assembled by Laura Gregorio in her essay The Smallpox Legacy, Pharos. Fall 1996

What is a vaccine?

Vaccines are harmless agents, perceived as enemies. They are molecules, usually but not necessarily proteins, that elicit an immune response, thereby providing protective immunity against a potential pathogen. While the pathogen can be a bacterium or even a eukaryotic protozoan, most successful vaccines have been raised against viruses and here we shall deal with anti-viral vaccines. Vaccines may consist of a purified protein, nucleic acid or a complex of molecules or even a whole bacterium or virus.

Immunity to a virus normally depends on the development of an immune response to antigens on the surface of a virally infected cell or on the surface of the virus particle itself. Immune responses to internal antigens often play little role in immunity. Thus, in influenza pandemics, a novel surface glycoprotein acquired as a result of antigenic shift characterizes the new virus strain against which the population has little or no immunity. This new strain of influenza virus may, nevertheless, contain internal proteins that have been in previous influenza strains. Surface glycoproteins are often referred to as protective antigens. To make a successful vaccine against a virus,  the nature of these surface antigens must  be known unless the empirical approach of yesteryear is to be followed. It should be noted, however, that a virally-infected cell displays fragments of internal virus antigens on its surface and these can elicit a cytotoxic T cell response that acts against the infected cell.

There may be more than one surface glycoprotein on a virus and one of these may be more important in the protective immune response than the others; this antigen must be identified for a logical vaccine that blocks infectivity. For example, influenza virus has a neuraminidase and a hemagglutinin on the surface of the virus particle.  It is the hemagglutinin that provokes neutralizing immunity because it is the protein that attaches the virus to a cell surface receptor and the neutralizing antibody interferes with virus binding to the cell.

In addition to blocking cell to virus attachment, other factors can be important in the neutralization of viruses; for example, complement can lyze enveloped virions after opsonization by anti-viral antibodies.

In this chapter, we deal mostly with anti-viral vaccines, although there are also successful anti-bacterial vaccines (see here).

WEB RESOURCES


Common Misconceptions about Vaccination and how to respond to them

Major sites of viral infection

In order to develop a successful vaccine, certain characteristics of the viral infection must be known. One of these is the site at which the virus enters the body. Three major sites may be defined:

• Infection via  mucosal surfaces of the respiratory tract and gastro-intestinal tract.

  • Virus families in this group are: rhinoviruses; myxoviruses; coronaviruses; parainfluenzaviruses; respiratory syncytial viruses; rotaviruses
     

• Infection via mucosal surfaces followed by spread systemically via the blood and/or neurones to target organs.

Virus families in this group are: picornaviruses; measles virus; mumps virus; herpes simplex virus; varicella virus; hepatitis A and B viruses

• Infection via needles or insect bites, followed by spread to target organs:

Virus families in this group are hepatitis B virus; alphaviruses; flaviviruses; bunyaviruses

IgA-mediated local immunity is very important in the first two categories. There is little point in having a good neutralizing humoral antibody in the circulation when the virus replicates, for example, in the upper respiratory tract. Clearly, here secreted antibodies are important.

Thus, we need to know:

• The viral antigen(s) that elicit neutralizing antibody

• The cell surface antigen(s) that elicit neutralizing antibody

• The site of replication of the virus

Types of vaccines

There are five basic types of vaccine in use today

• Killed vaccines: These are preparations of the normal (wild type) infectious, pathogenic virus that has been rendered non-pathogenic, usually by chemical treatment such as with formalin that cross-links viral proteins.

• Attenuated vaccines: These are live virus particles that grow in the vaccine recipient but do not cause disease because the vaccine virus has been altered (mutated) to a non-pathogenic form; for example, its tropism has been altered so that it no longer grows at a site that can cause disease.

• Sub-unit vaccines: These are purified components of the virus, such as a surface antigen.

• DNA vaccines: These are usually harmless viruses into which a gene for a protective antigen has been spliced. The protective antigen is then made in the vaccine recipient to elicit an immune response

• mRNA vaccines: These are the RNA-coding sequence of a protective (usually surface) antigen that is translated by the cells of the vaccinee after injection and expressed on the surface of transfected cells.

Problems in vaccine development

There are many problems inherent in developing a good protective anti-viral vaccine. Among these are:

• Different types of virus may cause similar diseases -- e.g. the common cold. As a result, a single vaccine will not be possible against such a disease

• Antigenic drift and shift -- This is especially true of RNA viruses and those with segmented genomes

• Large animal reservoirs. If these occur, re-infection after elimination from the human population may occur

• Integration of viral DNA. Vaccines will not work on latent virions unless they express antigens on the cell surface. In addition, if the vaccine virus integrates into host cell chromosomes, it may cause problems (This is, for example, a problem with the possible use of anti-HIV vaccines based on attenuated virus strains)

• Transmission from cell to cell via syncytia - This is a problem for potential AIDS vaccines since the virus may spread from cell to cell without the virus entering the circulation.

• Recombination and mutation of the vaccine virus in an attenuated vaccine.

Despite these problems, anti-viral vaccines have, in some cases, been spectacularly successful (see addendum) leading in one case (smallpox) to the elimination of the disease from the human population. The smallpox vaccine is an example of an attenuated vaccine, although not of the original pathogenic smallpox virus. Another successful vaccine is the polio vaccine which may lead to the elimination of this disease from the human population soon. This vaccine comes in two forms. The Salk vaccine is a killed virus, while that developed by Albert Sabin is a live attenuated virus vaccine. Polio is presently restricted to south Asia (Pakistan and Afghanistan).

Although smallpox is the only human disease that has been eradicated using vaccination, it is likely that one animal disease has also been eradicated. Rinderpest (cattle plague or steppe murrain) is a viral disease with high mortality that infects cattle and other ruminants and causes fever, diarrhea and lymphoid necrosis. It is a member of the measles family (Family: Paramyxoviridae; Genus: Morbillivirus) and was eradicated using a live attenuated vaccine. In 2010, the Food and Agriculture Organization reported that no case of rinderpest had been diagnosed for nine years. It is thus the only disease of agricultural livestock that has been successfully eradicated.

The work of art depicted in this image and the reproduction thereof are in the public domain worldwide. The reproduction is part of a collection of reproductions compiled by The Yorck Project. The compilation copyright is held by Zenodot Verlagsgesellschaft mbH and licensed under the GNU Free Documentation License.

Smallpox (Variola)

Smallpox is a devastating and disfiguring disease that is highly infectious. It is caused by variola virus (also known as smallpox virus), a member of the orthopoxvirdae (figure 2A). The disease of smallpox has been known for thousands of years and probably originated in Asia. It spread westwards into the middle east and among its victims was Pharaoh Rameses V (figure 2B). The disease may have reached Europe with the crusaders. Smallpox was introduced to the New World by European colonists and caused devastating epidemics in the indigenous population who had no natural immunity. Indeed, some early colonists used smallpox as a biological weapon again the original inhabitants of North and South America.

Smallpox is characterized by numerous pustules containing infectious virus all over the body (figure 2 C and D). The fatality rate is more than one quarter of infected patients infected by the most serious form caused by Variola major. Another form of smallpox caused by Variola minor has a much lower fatality rate (up to 5%).

The first attempts to control smallpox occurred in the 10th century and used variolation (so called because small pox virus is Variola). In variolation (figure 2E), material (scabs) was obtained from the pustules of an infected person who did not die of the disease. This person, therefore, had a milder form of smallpox as a result of a naturally occurring variant. This material was used to infect another person who usually also got a milder disease. If the person did not die, there was lifelong immunity.  Another reason for the success of variolation was that virus in the scabs was less virulent because it had been partially desiccated and was complexed with and inactivated by antibodies from the donor.

The fatality rate of variolation was about 1 - 2% and so it was still a dangerous procedure. This technique was used in Pakistan, Ethiopia and Afghanistan until 1970. Variolation was widespread in England in 1700s where it was introduced by the wife of the British Ambassador to Turkey, Mary Wortley Montague (figure 1b).

A. Smallpox virus
 Copyright 
1994 Veterinary Sciences 
Division  Queen's University Belfast

B. The mummified head of  Ramses V (died 1157 BCE)  with rash that is probably the result  of smallpox

 

   C. Infant with smallpox 

   D. Smallpox lesions on skin of trunk. Photo taken in Bangladesh. CDC/James Hicks 

E. Powdered smallpox scabs were inhaled to protect against smallpox in Chinese medicine

 
A. Edward Jenner

B. Dr Jenner about to vaccinate a child

 

 C.  Blossom the cow

D. The last known person in the world to have a natural case of smallpox. Variola minor in 23-year-old Ali Maow Maalin, Merka, Somalia
CDC

In 1796, Edward Jenner (figure 3A), who was at the time experimenting with variolation, discovered vaccination using vaccinia virus, the agent of cowpox (vacca is the Latin for cow). 

Jenner was a physician living in rural Gloucestershire in the west of England and it was widely known at that time that people who contracted cowpox (such as dairy maids) appeared to gain protective immunity against the much more virulent smallpox. Jenner vaccinated a Mr Phipps (who worked for him) and own son (figure 3B) with cowpox from a cow called Blossom (figure 3C), and then challenged them with virulent smallpox. Both vaccinees were, fortunately, protected. Jenner's original virus is not the vaccinia that was used in smallpox vaccinations until recently. The vaccine virus may have arisen as recombinant from cowpox or horse pox. For a long time  the vaccine virus was maintained in horses or buffalo.

The last case of natural smallpox in the U.K. occurred in the 1930s; the last in the U.S.A. was in the 1940s. The last natural case in the world was in Somalia and occurred in October 1977 (figure 3D). Although the virus had been eliminated in the wild, smallpox was retained in  laboratories and as a result of a laboratory accident there was a fatal case in Birmingham, England in 1978 when a medical photographer died. This person was the last person to die of smallpox in the world.

Worldwide stocks were reduced to laboratories in the United States and the Soviet Union. It is not known whether infectious virus from the Russian laboratory was distributed after the dissolution of the Soviet Union.

The eradication of smallpox has been one of the great triumphs of public health. There are several reasons for this:

• There is no animal reservoir for variola, only humans are infected by this virus

• Once a person has been infected by the virus, there is lifelong immunity, although this may not be the case with people immunized using the vaccine strain

• Subclinical cases are rare and so an infected person can be identified and isolated

• Infectivity does not precede overt symptoms, that is there is no prodromal phase

• There is only one Variola serotype and so the vaccine is effective against all virus strains

• The vaccine is very effective

• There has been a major commitment by the World Health Organization and governments to smallpox erradication.

Figure 5.
Rabies virus

Rabies

Almost a century after Jenner's pioneering work on smallpox, in 1885 Louis Pasteur (figure 4) and Emile Roux developed the first  vaccine against rabies (figure 5) (rabhas, Sanskrit: to do violence). Pasteur discovered that if he took spinal cord material from a dead rabid rabbit and kept it for a period of 15 days in a dry atmosphere (a flask containing potassium hydroxide) and then injected it into a dog, the latter did not get rabies. He developed a protocol in which he carried out the same procedure with spinal cord tissue that had been kept in a dry atmosphere for less and less time (each was separated by an interval of two days), until he finally injected spinal tissue containing virulent virus (only a day or two in the flask). He found that the dog was then immune to rabies. Pasteur successfully treated a boy (Joseph Meister)  bitten by a rabid dog sixty hours earlier with this protocol in which he used successively more virulent virus. In fact, according to Pasteur, the rabies in the final inoculation was more virulent than that of ordinary canine rabies. Fortunately, Mr Meister survived both the initial bites and the virulent virus!

Current anti-rabies vaccines are not prepared in the way that Pasteur used. Human Diploid Cell Vaccine (HDCV) is made in tissue culture using normal human WI-38 fibroblasts. The rabies virus is purified by passage through a filter and inactivated by beta-propriolactone. This inactivated virus vaccine is used almost exclusively in the developed world for pre- and post-vaccination of rabies. Purified Chick Embryo Vaccine (PCEC) is also purified virulent virus. It is made by ultracentrifugation and also inactivated by beta-propriolactone. These vaccines give a high titer of neutralizing antibody after 10 days. When used properly, they can confer 100% protection.

There is also a live attenuated vaccine (Flury strain) that is grown in chick embryos and is for use in animals only.

A recombinant anti-rabies vaccine (VRG, Raboral) has been made by inserting the gene for the surface glycoprotein of rabies into vaccinia virus, the virus used in smallpox vaccinations. The recombined virus appears safe for humans but is used for treating wild animals since (because it is a live virus) it can give herd immunity. The vaccine virus is stable to elevated temperatures and can be delivered orally. It is therefore fed to animals in food baits. Raboral V-RG® is approved for immunization of raccoons and coyotes, two of the most significant wildlife carriers of rabies in North America.

 

Poliomyelitis

In western countries, wild type polio is no longer a problem but it is still endemic in Pakistan and Afghanistan (figures 6). However, wild poliovirus has been imported into some countries that have stopped transmission of indigenous virus and  outbreaks can result from these importations. A number of countries continue to be affected by such outbreaks. Most of these are in the “wild poliovirus importation belt” – a number of countries stretching from west Africa to central Africa and the Horn of Africa (figure 6).

Until the 1950s, when anti-polio vaccination became routine, summer outbreaks of polio were common in western countries, often spread via the oral-fecal route while using swimming pools. These outbreaks led to widespread paralytic polio that necessitated help in breathing and the use of "iron lungs" (figure 7).

 

Anti-polio vaccines

There are two types of polio vaccine, both of which were developed in the 1950s. The first, developed by Jonas Salk, is a formalin-killed preparation of normal wild type polio virus. This is grown in monkey kidney cells and the vaccine is given by injection. It elicits good humoral (IgG) immunity and prevents transport of the virus to the neurons where it would otherwise cause paralytic polio. This vaccine is the only one used in some Scandinavian countries where it completely wiped out the disease.

A second vaccine was developed by Albert Sabin. This is a live attenuated vaccine that was produced empirically by serial passage of the virus in cell culture. This resulted in the selection of a mutated virus that grew well in culture and, indeed, in the human gut where the wild type virus grows. It cannot, however, migrate to the neurones. It replicates a normal infection since the virus actually grows in the vaccinee and it elicits both humoral and cell-mediated immunity. It is given orally, a route that is taken by the virus in a normal infection since the virus is passed from human to human by the oral-fecal route. This  became the preferred vaccine in the United States, United Kingdom and many other countries because of it ease of administration (often on a sugar lump), the fact that the vaccine virus replicates in the gut, and only one administration is needed to get good immunity (though repeated administration was usually used). In addition, the immunity that results from the Sabin vaccine lasts much longer that that by the Salk vaccine, making fewer boosters necessary. Since it elicits mucosal immunity (IgA) in the gut (figure 10), the Sabin vaccine has the potential to wipe out wild type virus whereas the Salk vaccine only stops the wild type virus getting to the neurons.

The attenuated Sabin vaccine, however, came with a problem: back mutation. This may result from recombination between wild type virus and the vaccine strain. Virulent virus is frequently isolated from recipients of the Sabin vaccine. The residual cases in countries that used the attenuated live virus vaccine (about 8 per year in the US until recently; figure 9a, b and c) resulted from mutation of the  vaccine strain to virulence. About half of these cases were in vaccinees and half in contacts of vaccinees. Paralytic polio arises in 1 in 100 cases of infection by wild type virus and 1 in 2.4 million initial vaccinations as a result of back reversion of the vaccine to virulence. This was deemed acceptable as the use of the attenuated virus means that the vaccine strain of the virus still replicates in the body and gives gut immunity via IgA.

The vaccinee who has received killed Salk vaccine still allows wild type virus to replicate in his/her gastro-intestinal tract, since the major immune response to the injected killed vaccine is circulatory IgG (figure 10). As noted above, this vaccine is protective against paralytic polio since, although the wild type virus can still replicate in the vaccinee's gut, it cannot move to the nervous system where the symptoms of polio are manifested. Thus, wild type virus is unlikely to die out in populations who have received only the killed vaccine since it would be shed in the feces. It should be noted, however, that studies in The Netherlands during a polio outbreak in 1992 (among people who had refused the vaccine) showed that immunity produced by the Salk vaccine did prevent circulation of wild type virus in the general population.

An additional problem of using a live attenuated vaccine is that preparations may contain other pathogens from the cells on which the virus was grown. This was certainly a problem initially because the monkey cells used to produce the polio vaccine were infected with simian virus 40 (SV40) and this was also in the vaccine. SV40 is a polyoma virus and has the potential to cause cancer. It appears, however, not to have caused problems in vaccinees who inadvertently received it. There have been some allegations that the original attenuated polio vaccine used in Africa may have been contaminated with human immunodeficiency virus (HIV). This has been found not to be the case. Of course, there can also be similar problems with the killed vaccine if it is improperly inactivated. This has also occurred.

Current recommendations concerning polio vaccines

Once the only polio cases in the US were vaccine-associated, the previous policy of solely using the Sabin vaccine was reevaluated. At first, both vaccines were recommended with the killed vaccine first and then the attenuated vaccine. The killed vaccine would stop the revertants of the live vaccine giving trouble by moving to the nervous system. Thus, in 1997 the following protocol was recommended:

To reduce the vaccine associated cases (8 to 10 per year), the CDC Advisory Committee on Immunization Practices (ACIP) has recommended (January 1997) a regimen of two doses of the injectable killed (inactivated: Salk) vaccine followed by two doses of the oral attenuated vaccine on a schedule of 2 months of age (inactivated), 4 months (inactivated), 12-18 months (oral) and 4-6 years (oral). Currently four doses of the oral vaccine are typically administered in the first two years of life. It is thought that the new schedule will eliminate most of the cases of vaccine-associated disease. This regimen has already been adopted by several European countries and some of Canada. 

The regimen of polio vaccination was subsequently amended again in 2000: 

To eliminate the risk for Vaccine-Associated Paralytic Poliomyelitis, the ACIP recommended an all-inactivated poliovirus vaccine (IPV) schedule for routine childhood polio vaccination in the United States. As of January 1, 2000, all children should receive four doses of IPV at ages 2 months, 4 months, 6-18 months, and 4-6 years.

In 2009, the recommendations were further revised:

Three different combination vaccines containing IPV have been licensed for routine use in the United States. Because of potential confusion in using different vaccine products for routine and catch-up immunization, ACIP recommends the following:
The 4-dose IPV series should continue to be administered at ages 2 months, 4 months, 6--18 months, and 4--6 years.
The final dose in the IPV series should be administered at age ≥4 years regardless of the number of previous doses.
The minimum interval from dose 3 to dose 4 is extended from 4 weeks to 6 months.
The minimum interval from dose 1 to dose 2, and from dose 2 to dose 3, remains 4 weeks.
The minimum age for dose 1 remains age 6 weeks.

 

CASE REPORT

Poliovirus Infections in Four Unvaccinated Children --- Minnesota, August--October 2005 

Polio - 1988 WHO
Polio - 1998 WHO

Polio - 2004
WHO

Polio - 2013
polioeradication.org

Polio 2020
Our World in data

A. Child in iron lung
WHO

B. Iron lung ward

C. Child with polio sequelae
WHO

D. Child with polio sequelae
WHO

E. Victims of polio
WHO  

  Figure 9a
Reported (cases per 100,000 population) cases of paralytic poliomyelitis in the United States, 1951-1992, which initially used the killed Salk vaccine. This was subsequently replaced by the live attenuated oral vaccine developed by Albert Sabin. The Sabin vaccine is swallowed. It is often given on a sugar lump

Figure 9b
Poliomyelitis in the US 1980-1995 CDC

  Figure 9c
Vaccine-associated paralytic polio - VAPP in US  1964-1995 CDC

Figure 10
 Secretory antibody (nasal and gut IgA) and serum antibody (serum IgG, IgM and IgA) in response to killed polio vaccine (left) administered by intramuscular injection and to live attenuated polio vaccine (right) administered orally 
 

Figure 10b
Countries with the highest numbers of rotavirus deaths in children
WHO

Figure 10c
Rotavirus mortality in children under five in 2013
WHO

Rotavirus disease: An initial problem but later success

Rotaviruses are found worldwide and almost very child in the world is infected by rotaviruses by the age of five. These viruses cause major gastroenteritis and diarrhea-associated hospitalization and over 215,000 deaths in 2013 in children under five years of age. In 2013, 37% of deaths in children resulted from diarrhea (figure 10a). The virus is spread by the oro-fecal route.

According to WHO, five countries (India, Nigeria, Pakistan, the Democratic Republic of the Congo and Angola) accounted for more than half of all rotavirus disease deaths under age five (figure 10b and c). Symptoms include: fever, vomiting, diarrhea and abdominal pain. Seroprevalence studies show that antibody is present in most infants by age 3 years.

Prior to the introduction in the United States of widespread vaccination in 2006, there were up to three million cases of rotavirus infection per year. In about 1 to 2.5% of cases, there was severe dehydration. This resulted in 20 to 60 deaths of children under five each year. In addition, there were 50,000 to 70,000 hospitalizations and over 500,000 visits to doctors’ offices per year.

Since the introduction of vaccination there has been a drop in rotavirus-related hospitalizations by up to 86 percent. It is likely that vaccination has also protected non-vaccinated infants by limiting circulating infection. Deaths have also been markedly reduced. In 2008, there were an estimated 14 deaths from rotavirus disease in the United States and fewer than 10 in the United Kingdom compared to 98,621 in India.

Rotashield and intussusception

Reassortant vaccines are created by genetic reassortment in which non-human rotavirus strains express the antigens of human rotaviruses on their surface. The non-human strains replicate but do not cause disease and are of low pathogenicity in humans. A live, tetravalent rhesus-human reassortant vaccine (Rotashield - Wyeth Laboratories) was first licensed for use in infants in August 1998. It contained human G types 1, 2, 4, and simian G type 3. However, post-licensure surveillance indicated a possible relationship between the occurrence of intussusception 3 to 20 days after the vaccine was administered, especially the first dose (15 cases/1.5 million doses were reported).  Intussusception is a rare intestinal obstruction that occurs when the intestine folds on itself or telescopes into itself resulting in reduced blood supply. It is most common among young children. The most common place in the intestine for intussusception to occur is where the small intestine joins the colon. However, it can occur in many parts of the intestine. With prompt treatment, almost all patients fully recover. It is more common in boys than in girls.

Use of the Rotashield vaccine was suspended and it was eventually removed from the market in October 1999, when studies confirmed the link between vaccination and intussusception.

RotaTeq

RotaTeq (Merck) is a live oral vaccine licensed in the United States in 2006. It contains five reassortants (WC3 bovine rotavirus strain with surface proteins of the G1-4 and P1A human serotypes). It does not contain preservatives or thimerosal. Three doses are given at 2, 4 and 6 months of age with the minimum age for the first dose of 6 weeks. The series should not be initiated after 12 weeks. The efficacy of the RotaTeq vaccine is high with 98% reduction in severe rotavirus gastroenteritis within the first year of vaccination and a 96% reduction in hospitalization rate. There is also a 74 and 71% reduction of rotavirus gastroenteritis within the first and second years after vaccination.

Rotarix

Rotarix (Glaxo Smith Klein) is a human, live attenuated rotavirus vaccine which contains a rotavirus strain of G1P[8] specificity. It is used for the prevention of rotavirus gastroenteritis caused by G1 and non-G1 types (G3, G4, and G9) when administered as a 2-dose series in infants and children.

Both of these rotavirus vaccines are very effective (85% to 98%) at preventing infection-associated gastroenteritis and diarrhea. CDC recommends routine vaccination of infants with either of the two available vaccines. Both are administered orally.

• RotaTeq® (RV5). This is given in 3 doses at ages 2 months, 4 months, and 6 months
• Rotarix® (RV1). This is given in 2 doses at ages 2 months and 4 months.

Other vaccines include Rotavac (India), Rotavin (Vietnam) and Langzhou Lamb (China).

 

OTHER ANTI-VIRAL VACCINES

There are a number of other commonly used anti-viral vaccines and these are listed below

TABLE 1 Common currently used anti-viral vaccines
Virus	Vaccine Type	Micrograph	CDC Links
Polio (Salk)	Inactivated	Transmission electron micrograph of poliovirus type 1. CDC/Dr. Joseph J. Esposito  jje1@cdc.gov	Updated Recommendations of the Advisory Committee on Immunization Practices (ACIP) Regarding Routine Poliovirus Vaccination (2009)
Polio (Sabin)	Attenuated
Rabies	Current human vaccine is inactivated. There is an attenuated vaccine for animal use.	Rabies Virus New York State Department of Health	ACIP Recommendations Use of a Reduced (4-Dose) Vaccine Schedule for Postexposure Prophylaxis to Prevent Human Rabies (2008)
Mumps	Attenuated	Mumps Virus CDC PHIL	Use of Combination Measles, Mumps, Rubella, and Varicella Vaccine Recommendations of the Advisory Committee on Immunization Practices (ACIP)
Measles	Attenuated	Measles Virus CDC PHIL
Rubella	Attenuated	Rubella virus CDC PHIL
Influenza	Inactivated	Influenza virus Copyright 1994 Veterinary Sciences Division  Queen's University Belfast	CDC Vaccine Information Prevention and Control of Influenza with Vaccines: Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2011
Hepatitis A	Inactivated		Prevention of Hepatitis A Through Active or Passive Immunization. Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2006
Hepatitis B	Subunit	Hepatitis B Virus Copyright Dr Linda M Stannard, University of Cape Town, South Africa, 1995.	Hepatitis B Vaccine Recommendations (2005, 2006, and 2011) Part 1 - Infants, Children, & Adolescents Part 2 - Adults
Varicella	Attenuated	Varicella Virus John Curtin School of Medical Research Australian National University Canberra, Australia. Micrograph:  Dr Frank Fenner	Prevention of Varicella Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2007
Rotavirus	Attenuated	Rotavirus  Copyright 1994 Veterinary Sciences Division , Queen's University, Belfast	Prevention of Rotavirus Gastroenteritis Among Infants and Children Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2009 Rotavirus and Intussusception
Yellow Fever	Attenuated	Yellow fever virus CDC PHIL	Yellow Fever Vaccine Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2010
Human Papilloma	Subunit		Quadrivalent Human Papillomavirus Vaccine Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2007
Japanese Encephalitis	Inactivated		Japanese Encephalitis Vaccines Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2010
Varicella	Attenuated	Varicella (Chickenpox) Virus CDC PHIL	Prevention of Varicella. Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2007
Herpes Zoster	Attenuated		Prevention of Herpes Zoster. Recommendations of the Advisory Committee on Immunization Practices (ACIP), 2008
For more information on anti-HIV (AIDS) vaccines go here

KILLED VERSUS ATTENUATED VACCINES

Attenuated Vaccines

Attenuation is usually achieved by passage of the virus in foreign host such as embryonated eggs or tissue culture cells. From among the many mutant viruses that exist in a population (especially so in RNA viruses), some will be selected  that have a better ability to grow in the foreign host (higher virulence). These tend to be less virulent for the original host. To produce the Sabin polio vaccine, attenuation was only achieved with high inocula and rapid passage in primary monkey kidney cells. The virus population became overgrown with a less virulent strain (for humans) that could grow well in non-nervous (kidney) tissue but not in the central nervous system. Non-virulent strains of all three polio types have been produced for the vaccine.

Molecular basis of attenuation

 We do not know  the basis of attenuation in most cases since attenuation was achieved empirically. The empirical foreign-cell passage method causes many mutations in a virus and it is difficult to determine which are the important mutations. Many attenuated viruses are temperature-sensitive (that is, they grow better at 32 - 35 degrees than 37 degrees) or cold adapted (they may grow at temperatures as low as 25 degrees). In the type 1 polio virus attenuated vaccine strain, there are 57 nucleotide changes in the genome, resulting in 21 amino acid changes . One third of the mutations are in the VP1 gene (this gene is only 12% of genome). This suggests that attenuation results from changes in surface proteins of the virus

An attenuated nasal vaccine for influenza (FluMist®) has been developed (see below). This contains cold-adapted vaccine strains of the influenza virus that have been grown in tissue culture at progressively lower temperatures. After a dozen or more of these passages, the virus grows well only at around 25° and in vivo growth is restricted to the upper respiratory tract. Studies showed that influenza illness occurred in only 7 percent of volunteers who received the intra-nasal influenza vaccine, versus 13 percent injected with trivalent inactivated influenza vaccine and 45 percent of volunteers who were given placebo. Both vaccine comparisons with placebo were statistically significant.

Advantages of attenuated vaccines

• They activate all phases of immune system. They elicit humoral IgG and local IgA (figure 8)

• They raise an immune response to all protective antigens. Inactivation, such as by formaldehyde in the case of the Salk vaccine, may alter antigenicity

• They offer more durable immunity and are more cross-reactive. Thus, they stimulate antibodies against multiple epitopes which are similar to those elicited by the wild type virus

• They cost less to produce

• They give quick immunity in majority of vaccinees

• In many cases (e.g. polio and adenovirus vaccines), administration is easy

• These vaccines are easily transported in the field

• They can lead to elimination of wild type virus from the community

Disadvantages of Attenuated vaccine

• Mutation. This may lead to reversion to virulence (this is a major disadvantage)

• Spread to contacts of the vaccinee who have not consented to be vaccinated (This could also be an advantage in communities where vaccination is not 100%)

• Spread of the vaccine virus that is not standardized and may be mutated

• Sometimes there is poor "take" in tropics

• Live viruses are a problem in immunodeficiency disease patients

Advantages of inactivated vaccine

• They give sufficient humoral immunity if boosters given

• There is no mutation or reversion (This is a big advantage)

• They can be used with immuno-deficient patients

• Sometimes they perform better in tropical areas

Disadvantages of inactivated vaccines

• Some vaccinees do not raise immunity

• Boosters tend to be needed

• There us little mucosal / local immunity (IgA). This is important (figure 8)

• Higher cost

• In the case of polio, there is a shortage of monkeys

• In the case of smallpox, there have been failures in inactivation leading to immunization with virulent virus.

New methods of vaccine production

Selection for mis-sense
Conditional lethal mutants. Temperature-sensitive mutants in influenza A and RSV have been made by mutation with 5-fluorouracil and then selected for temperature sensitivity. In the case of influenza, the temperature-sensitive gene can be reassorted in the laboratory to yield a virus strain with the coat of the strains circulating in the population and the inner proteins of the attenuated strain.  Cold adapted mutants can also be produced in this way. It has been possible to obtain mis-sense mutations in all six genes for non-surface proteins.

The attenuated influenza vaccine, called FluMist, uses a cold-sensitive mutant that can be reassorted with any new virulent influenza strain that appears (figure  11). The reassorted virus will have the genes for the internal proteins from the attenuated virus (and hence will be attenuated) but will display the surface proteins of the new virulent antigenic variant. Because this is based on a live, attenuated virus, the customization of the vaccine to each year's new flu variants is much more rapid than the process of predicting what influenza strains will be important for the coming flu season and combining these in a killed vaccine.

Synthetic peptides
Injected peptides which are much smaller than the original virus protein raise an IgG response but there is a problem with poor antigenicity.  This is because the epitope may depend on the conformation of the protein in the virus as a whole. A non-viral example that has achieved some limited success is a prototype anti-malarial vaccine. 

Anti-idiotype vaccines
An antigen binding site in an antibody is a reflection of the three-dimensional structure of part of the antigen, that is of a particular epitope. This unique amino acid structure in the antibody is known as the idiotype which can be thought of as a mirror of the epitope in the antigen. Antibodies (anti-ids) can be raised against the idiotype by injecting the antibody into another animal. This gives us an anti-idiotype antibody and this, therefore, mimics part of the three dimensional structure of the antigen, that is, the epitope (figure 12). This can be used as a vaccine. When the anti-idiotype antibody is injected into a vaccinee, antibodies (anti-anti-idiotype antibodies) are formed that recognize a structure similar to part of the virus and might potentially neutralize the virus. This happens: Anti-ids raised against antibodies to hepatitis B S antigen elicit anti-viral antibodies.

Recombinant DNA techniques

Attenuation of virus
Deletion mutations can be made that are large enough that they are unlikely to revert (though suppression of the mutation remains a problem. This has been seen in some of the Nef deletion mutants developed as potential anti-HIV vaccines). Another problem with this approach in some vaccines is that the virus could still retain other unwanted characteristics such as oncogenicity (e.g. with adenovirus, herpes virus, HIV). 

Single gene approach (usually a surface glycoprotein of the virus)
A single gene (for a protective antigen) can be expressed in a foreign host. Expression vectors are used to make large amounts of antigen to be used as a vaccine. The gene could be expressed in and the protein purified from bacteria using a fermentation process, although lack of post-translational processing by the bacteria is a problem. Yeast are better for making large amounts of antigen for vaccines since they process glycoproteins in their Golgi bodies in a manner more similar to mammals. An example of a vaccine in which a viral protein is expressed in and purified from yeast is Gardasil, an anti-human papilloma virus vaccine that is very effective in preventing cervical cancer. The current hepatitis B vaccine is also this type. A similar anti-human papilloma vaccine, Cervarix, is made by expressing viral genes recombined into a bacculovirus and expressed in insect cells.

Expression of the SARS-CoV-2 S protein in bacculovirus is also used in the preparation of the Novavax COVID-19 vaccine (see below) which has been shown to be highly effective, possibly because of the use of an effective proprietary adjuvant.

These vaccines have many of the disadvantages of a killed vaccine. This approach has been used to make several potential HIV vaccines but they provoke little cell-mediated immunity.

Cloning of a gene into another virus
By cloning the gene for a protective antigen into different harmless virus, the antigen is presented just as in the original virus. In addition, cells become infected, leading to cell-mediated immunity. Vaccinia (the smallpox vaccine virus) is a good candidate since it has been widely used in the human population with no ill effects. Moreover, a multivalent vaccine virus strain can be made in this way as vaccinia will accept several foreign genes. A candidate HIV vaccine has undergone clinical trials. However, the use of vaccinia against smallpox has shown rare but serious complications in immuno-compromised patients and alternatives have been sought. One is a recombinant canary pox virus that does not replicate in humans but does infect cells. Immunization with live recombinant canary pox vector expressing the HIV envelope gene has induced an HIV-1 envelope specific CTL response. Similar constructs with gag, protease, nef and parts of pol genes have been studied in clinical trials but all have, so far, shown no clinical efficacy.

Today the most widely used virus vector is a modified human or chimpanzee adenovirus which have been used in several COVID-19 vaccines.

DNA Vaccines

These vaccines are based on the introduction of a DNA plasmid or whole virus into the vaccinee. The vaccine carries an extra protein-coding gene that expresses an antigen that causes an immune response; for example, some COVID-19 vaccines use a replication-deficient adenovirus expressing the SARS-CoV-2 S protein.

These vaccines are often called DNA vaccines but would better be called DNA-mediated or DNA-based immunization since it is not the purpose to raise antibodies against the DNA molecules themselves but to get the protein expressed by cells of the vaccinee. Usually, muscle cells do this since the DNA is injected intramuscularly.

WEB RESOURCES
DNA vaccine web
 

Advantages of DNA vaccines

• Plasmids or DNA viruses are easily manufactured in large amounts

• DNA is very stable

• DNA resists temperature extremes and so storage and transport are straight forward

• A DNA sequence can be changed easily in the laboratory. This means that we can respond rapidly to variants in the infectious agent. This has become particularly important in developing vaccines against COVID-19.

• By using DNA injected into the vaccinee to code for antigen synthesis, the antigenic protein(s) that are produced are processed (post-translationally modified) in the same way as the proteins of the virus against which protection is to be produced. This makes a far better antigen than, for example, using a recombinant plasmid to produce an antigen in yeast (e.g. the HBV vaccine), purifying that protein and using it as an immunogen.

• Mixtures of DNA constructs could be used that encode many protein fragments from a virus or viruses so that a broad spectrum vaccine could be produced

• The DNA construct does not replicate in the vaccinee and encodes only the proteins of interest

• Because of the way the antigen is presented, there is a cell-mediated response that may be directed against any antigen in the pathogen. This also offers protection against diseases caused by certain obligate intracellular pathogens (e.g. Mycobacterium tuberculosis)

All of the above means that DNA vaccines are cheap and therefore likely to be developed against pathogens of lesser economic importance (at least to drug companies)

Possible Problems

• Potential integration of DNA into the host genome leading to insertional mutagenesis

• Induction of autoimmune responses (e.g. pathogenic anti-DNA antibodies)

• Induction of immunologic tolerance (e.g. where the expression of the antigen in the host may lead to specific non-responsiveness to that antigen)

 

The current influenza vaccine is an inactivated preparation containing antigens from the flu strains that are predicted to infect during the next flu season. If such a prediction goes awry, the vaccine is of little use. It is the surface antigens that change as a result of reassortment of the virus in the animal (duck) reservoir (see influenza). The vaccine is injected intramuscularly and elicits an IgG response (humoral antibody in the circulation). The vaccine is protective because enough of the IgG gets across the mucosa of the lungs where it can bind and neutralize incoming virus by binding to surface antigens. If a DNA vaccine is used, both humoral and cytotoxic T lymphocytes (CTL) are produced, which recognize antigens presented by vaccine-transfected cells. The CTLs are produced because the infected muscle cells present flu antigens in association with MHC class I molecules. If the antigen presented is the nucleocapsid protein (which is a conserved protein), this overcomes the problem of antigenic variation. Such an approach could revolutionize the influenza vaccine.

Other studies have used a mix of plasmids encoding both nucleoprotein and surface antigens. Protection by DNA vaccines has also been demonstrated with rabies, mycoplasma and Plasmodium yoelii.  Anti-HIV vaccines are also being tested. In the HIV chapters, it was noted that progress on AIDS vaccines has been stymied by the fact that many vaccines only elicit humoral antibodies while the use of whole virus vaccines (which might elicit CTL responses) has been rejected because of other potential problems. Plasmid-based vaccines may overcome these problems

 

ADENOVIRUS-BASED DNA VACCINES

AstraZenica/Oxford University Vaccine: AZD1222, CHADOX1 NCOV-19

The ChAdOx1 nCoV-19 vaccine (AZD1222) consists of the replication-deficient simian adenovirus vector ChAdOx1, containing the full-length S glycoprotein gene of SARS-CoV-2, with a tissue plasminogen activator leader sequence. ChAdOx1 nCoV-19 expresses a codon-optimized coding sequence for the S protein. A simian adenovirus rather than a human one is used because the use of human adenovirus is limited by pre-existing immunity to the virus within the human population that significantly reduces the immunogenicity of vaccines based on the human virus. This is not a problem with the simian virus because, although simian adenoviruses are closely related to human adenoviruses, the hypervariable regions of the main immunogen are significantly different from the human virus thus avoiding preexisting immunity.

The simian adenovirus vectors lack the E1 region encoding viral transactivator proteins which are essential for virus replication and the E3 region encoding immunomodulatory proteins. The latter deletion allows incorporation of larger genetic sequences into the viral vector.

The vaccine adenovirus is taken up by cells and is transcribed in the nucleus to give mRNA which is translated to S protein.

Efficacy is up to 90%, depending on the dosage. Higher efficacy was found in a subgroup in which the first of two doses was halved. The average efficacy was 70.4%.

AD5-NCOV, Convidicea (Cansino Biologics, China)

This is another adenovirus-based vaccine. It is based on recombinant replication-defective human adenovirus type-5 vector to induce an immune response. Again, the virus has been rendered replication-deficient by deletion of the E1 and E3 genes. It encodes an optimized full-length S protein gene based on Wuhan-Hu-1 virus sequence with the tissue plasminogen activator signal peptide gene.

GAM-COVID-VAC, Sputnick V (Gamaleya Research Institute of Epidemiology and Microbiology, Russia)

Gam-COVID-Vac is a two-vector vaccine based on two modified human adenoviruses containing the gene that encodes the S protein of SARS-CoV-2. The first inoculation uses adenovirus 26 (Ad26) as the vector for the S protein gene while the second uses adenovirus 5 (Ad5). This vaccine was shown in January, 2021, to have 91.6% efficacy against symptomatic Covid-19.

AD26.COV2.S, JNJ-78436735 (Janssen/Johnson and Johnson, United States and Belgium)
This vaccine is based again on a recombinant modified adenovirus vector. Like the Sputnick vaccine, it uses human Ad26 expressing the S protein, in this case in a single inoculation. It raises a strong neutralizing antibody and cell-mediated response. It uses AdVac technology which increases stability so that the vaccine may be stored at refrigerator temperatures for at least three months.

MRNA VACCINES

The first two vaccines for COVID-19 approved in late 2020 were based on a protocol in which mRNA coding for the antigen of interest surrounded by a lipid carrier (lipid nanoparticle) is injected into the vacinee. The lipid protects the mRNA from ribonucleases and facilitates its entry into cells. The mRNA is translated to protein, processed and presented to the immune system in the usual way. The protein of interest is usually that which binds to the cell receptor and antibodies to this protein which block virus-cell receptor interaction will prevent infection and are called neutralizing antibodies. In the case of vaccines against SARS-CoV-2, this is the S antigen that binds to the human ACE2 receptor.

A major problem with mRNA vaccines is their stability in transit from the site of manufacture, outside the cell at the site of injection and within the cell. DNA Is inherently stable within the cell since it must pass the genetic code from cell to cell indefinitely. In contrast, mRNAs have a very short life compared to DNA. The amount of a mRNA depends on the balance between the rate of synthesis and the rate of degradation. Many proteins are required only for a very short time, and if their mRNAs were very stable the protein level could not be controlled. Hence, although all mRNAs have short lives, many are degraded very rapidly after translation, facilitating rapid responses to the conditions in the cell. The mRNAs are degraded by ribonucleases (RNAses). Different mRNAs have different degrees of stability resulting from their secondary structure and the nature of the ends of molecule. These are known as cis elements. In addition, their stability is also regulated by RNA-binding factors or trans elements. Cis elements include the 3’ poly A tail and the 5’ methyl guanosine cap. The 3’ poly A tail is bound by poly A-binding proteins that stabilize the RNA. These proteins require a certain length of poly A tail to bind and so the longer the poly A tail, the more of these proteins can bind to the RNA. mRNA is degraded from the 3’ end by 3’-5’ exonucleases and the 5’ end by removal of the 5’ cap and 5’-3’ exonuclease activity. Endonuclease activity also degrades mRNA and this can be regulated by other RNA binding proteins. AU-rich sequences in the 3’ untranslated region (UTR) are also involved in stability.

MRNA may also be stabilized by chemical modification of the bases of the nucleic acid itself. Such modifications include methyl adenosine, N-1-methylpseudouridine and pseudouridine (made from uridine by pseudouridine synthase (figure 13)), a base modification that is common in tRNA and enhances its stability. In mRNA these substituted bases enhance translation. Pseudouridine and N-1-methylpseudouridine repress intracellular signaling triggers for protein kinase R activation which is involved in mRNA stability. Of course, such modifications must not alter the fidelity of the translation of the message.

MRNA vaccines are made by the transcription of a plasmid encoding a protein recognized by a neutralizing antibody, in the case of a Covid-19 vaccine, this is the S protein. The plasmid, which contains the appropriate promoter sequences, is linearized and transcribed in vitro using a T7, T3 or Sp6 phage RNA polymerase. The resulting product contains an open reading frame that encodes the S protein flanked by 5’ and 3’ UTRs, a 5’ methyl guanosine cap and a poly A tail. This is what is used as the vaccine.

Figure 14 shows one way that this might be done in a system from AmpTec. The S protein gene is cloned into an insertion site in an m13 plasmid along with a T7 promotor (A). A forward primer complementary to the end of the M13 sequence (Pri) and a second reverse primer complementary to the end of the S gene are used (B). The latter primer includes a poly T sequence, usually around 120 nucleotides which does not hybridize to any m13 sequence. Using PCR, the DNA structure shown in C is produced. This is then used in in vitro transcription from the T7 promoter to form the polyadenylated mRNA shown in D. In vitro transcription can be carried out in the presence of modified nucleotides such as pseudouracil and/or N6-methyl adenosine, 5-methyl cytidine and others. These modified mRNAs are much more stable than normal mRNAs and are highly translatable giving the vaccine much increased efficacy.

The resultant protein is processed in the normal way through the exocytic pathway with all the usual post-translational modifications including glycosylation and transported to the cell surface. As described above, the protein may also be cleaved by proteases to form small peptides that can be presented at the cell surface to the immune system. The cell has anti-viral mechanisms to detect and degrade foreign RNAs and steps are taken to minimize this.

Even with nucleotide modifications, naked mRNA is likely to be rapidly degraded when injected into the vaccinee. In addition, the mRNA must cross the cell membrane to gain access to the cell protein translation machinery. Both of these problems can be solved by encapsulating the mRNA in a lipid envelope (a lipid nanoparticle or liposome) that helps the mRNA vaccine enter the cytoplasm from the endosome before it is degraded in a lysosome.

The initial Covid mRNA vaccines from BioNtec and Moderna use a technology similar to the above. A modification that may well be used in future mRNA vaccines is to make an mRNA vaccine which contains not only mRNA for the protein of choice (e.g. the SARS-CoV-2 S protein) but also mRNA for a viral RNA-dependent RNA polymerase (replicase). When this type of mRNA vaccine is injected into a vaccinee and enters a cell, it will be translated into S protein and into the replicase (which may be encoded on the same mRNA or a second mRNA). The viral replicase can recognize viral replication signals included in the vaccine mRNA(s) and can then amplify the input vaccine mRNA, making more copies of the mRNA and therefore more of the protein. Since there is now more of the vaccine mRNA in the cell than was originally delivered to the cytoplasm, this is known as the self-amplifying (SA) mRNA approach.
 

Tozinameran (BNT162B2. Brand name: Comirnaty) Pfizer-BioNTech Covid-19 vaccine

Tozinameran was the first mRNA vaccine to be approved. In clinical trials its efficacy is around 95%, 28 days after the first dose and is well tolerated. In one of the initial trials, there were 170 confirmed cases of Covid-19 of which 162 were in the placebo group and only 8 in the vaccine group. It is given in two doses, three weeks apart. It was not evaluated for asymptomatic infection. It appears to be effective against the variants described above. This vaccine must be stored and transported at -70 C.

It contains (WHO Non-proprietary names Program):

A modified 5’-cap1 structure (m7G+m3'-5'-ppp-5'-Am)

5´-untranslated region derived from human alpha-globin RNA with an optimized Kozak sequence. The latter ensures that the protein is correctly translated by the ribosome and functions as the translation initiation site in most eukaryotic mRNAs.

S glycoprotein signal peptide necessary for directing the nascent protein/ribosome complex to the signal receptor on the cytoplasmic surface of the rough endoplasmic reticulum membrane. This guides protein translocation to the correct orientation in the endoplasmic reticulum.

Codon-optimized sequence encoding full-length SARS-CoV2 S protein that contains two mutations: K986P and V987P. These alter the folding of the S protein so that it adopts an antigenically optimal pre-fusion conformation. All of the uridines are replaced by 1- methyl-3’-pseudouridine residues (Ψ) (figure 15) that are nevertheless efficiently translated.

At the end of the coding sequence are two ΨGA stop codons

The 3´ untranslated region comprises two sequence elements that confer RNA stability and high protein expression.

A 110-nucleotide poly A-tail consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another 70 adenosine residues.

In addition the vaccine contains lipids that make up the solid lipid nanoparticles that encapsulate the mRNA (ALC-0315 = ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; 1,2-Distearoyl-sn-glycero-3-phosphocholine; and cholesterol. In addition, the vaccine contains water, sucrose, dibasic sodium phosphate dehydrate, monobasic potassium phosphate, potassium chloride and sodium chloride.

Moderna Vaccine. mRNA1273

The Moderna vaccine is also an mRNA consisting of a synthetic message encoding the pre-fusion stabilized spike glycoprotein of SARS-CoV-2 virus. Pre-fusion stabilization is achieved by the substitution of two prolines as in the BioNTech vaccine.

Again, the mRNA is made by transcription from a T7 promotor in a reaction in which UTP was substituted with 1-methylpseudoUTP. In addition to the mRNA, the vaccine contains lipids to form a lipid nanoparticle: (SM-102, 1,2-dimyristoyl-rac-glycero3-methoxypolyethylene glycol-2000 [PEG2000-DMG], cholesterol, and 1,2-distearoyl-snglycero-3-phosphocholine) and, tromethamine, tromethamine hydrochloride, acetic acid, sodium acetate, sucrose and water.

The efficacy of m1273 is around 94.1%, similar to the BioNTec vaccine. In an initial trial, there were 196 confirmed cases of Covid-19 of which 185 were in the placebo group and 11 in the vaccine group.

It has the advantage over the latter in that the different lipid nanoparticle formulation allows it to be stored and transported at 2-8C, rather than the -70C of the BioNTec vaccine. It is administered in two doses, three weeks apart.

 

OTHER COVID-19 VACCINES

In the race to develop vaccines for COVID-19, many older strategies that have shown success in the past have also been used. These include subunit vaccines, an approach similar to that used to develop the highly successful vaccines for hepatitis B, and inactivated virus particles, an approach first used for the Salk polio vaccine.

Subunit vaccine: NVX-CoV2373, Novavax
The Novavax vaccine (NVX-CoV2373) is based on older technology using purified SARS-CoV-2 S protein with a Matrix M  adjuvant. In clinical trials, it produced high levels of anti-S protein antibodies and has been ordered by several governments as part of their anti-Covid-19 strategy. The gene for the S protein is inserted into a baculovirus. The Baculoviridae are a family of double-stranded circular DNA (80-180 base pairs) viruses that infect insects and arthropods. The modified baculovirus is then use to infect insect cells (usually Sf9 cells, isolated from Spodoptera frugiperda, the fall army worm) which make the S protein. This assembles into native trimers on the surface of the infected cell. These proteins are extracted and associated with lipid nanoparticles so that the S protein is presented to the immune system in a manner similar to that on the surface of an infected cell. Included with the vaccine is an adjuvant extracted from Quillaja saponaria, the soap bark tree (which, as its name implies can be used as a soap). In the case of vaccines, it stimulates the attraction of immune cells to the site of the injection where they respond more effectively. The adjuvant properties come from saponins (triterpene glycosides). The nanoparticles containing the S protein are taken up by antigen-presenting cells, cleaved into peptides and presented on the cell surface in association with MHC antigens to T and B cells.

Phase 3 trials have shown that this vaccine has 89% efficacy against Covid-19 and appears to provide strong immunity against the UK and South African variants.

Inactivated virus particles: Valneva vaccine (VLA 2001)
This uses an even more established vaccine technology similar to that used in the Salk polio vaccine, that is the use of inactivated whole virus particles. Virus is grown on African Green Money Kidney (Vero) cells, purified and inactivated with an agent such as formalin. The vaccine also contains alum and CpG 1018 adjuvants. CpG 1018 is a toll-like receptor 9 (TLR9) agonist.

There are a number of other SARS-CoV-2 vaccines in phase I and II trials that use formalin-inactivated whole virus particles (Sinovac and Sinopharm)

 

WHY DO WE NEED TWO INOCULATIONS?

Most of the vaccines that have been developed against SARS-CoV-2 require two inoculations. This is because of the way that the immune system responds to a foreign pathogen such as an infecting virus.

Initially, it is important to suppress infection by stopping the invading pathogen entering cells and replicating. Infection by a virus binding to its receptor on the cell surface (ACE2 in the case of SARS-CoV-2) triggers an initial response in which plasma B lymphocytes produce neutralizing antibodies that bind to the surface of the invading organism thereby, in the case of SARS-CoV-2, blocking virus S protein binding to ACE2. The initial antibody response, however, quickly declines but some of the B cells differentiate into memory B cells that survive for a long time and relocate to the periphery of the body. Here, they will be more likely to encounter more antigen during a second exposure. When this happens, they proliferate and differentiate into more plasma B cells, which then respond to the antigen by producing more antibodies. Memory B cells can survive for many years so that they are able to respond to multiple exposures to the same antigen.

During the first phase of the immune response, the immune cells also secrete cytokines that recruit other immune cells to the site of infection, among which are CD4-positive helper and cytotoxic T cells (killer T cells) that recognize and kill virus-infected cells. As with B cells, some T cells differentiate into memory cells that can reactivate and proliferate in response to new exposure to the original antigen. These memory T cells can also remain in the body for many years (and perhaps for a lifetime).

Since only a small number of memory T cells are made as a result of the initial exposure, a second exposure to the antigen (infection or inoculation) is required to boost their levels. Thus, with the mRNA SARS-CoV-2 vaccines, protection starts about 12 days after the first inoculation and rises to about 50% effectiveness. After a second injection three to four weeks later, the second phase of the immune response starts, memory B and T cells increase and effectiveness rises to around 95%.

  Figure 13
Pseudouridine and uridine structure

  Figure 14
Transcription of an mRNA vaccine molecule from a DNA plasmid construct

  Figure 15
1-methylpseudouridine. An extra methyl group is added enzymatically to the base of the pseudouracil

Today, many anti-viral vaccines are available and more are being developed. These vaccines have made a considerable impact on public health around the world (figure 13 and and see here).

WEB RESOURCES
Baseline 20th century annual morbidity and 1998 provisional morbidity from nine diseases with vaccines recommended before 1990 for universal use in children in the United States
MMWR/CDC

 

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