BIOLOGICS

BIOLOGICS

Introduction

Vaccines Toxins Toxoids Antitoxins Venoms Antivenins Antiserums Plant-based biologics

INTRODUCTION
Biologics means any product derived from a living plant or animal source.

However, strictly interpreted, the term biologics refers to “any virus, therapeutic serum, toxin, antitoxin or
analogous product,” and it has been interpreted to include a lengthy list of such products as vaccines of
bacterial, rickettsial, and viral origin, immune serums for the prevention or treatment of disease, various
miscellaneous and diagnostic products, human blood, and products derived from human blood.

Such substance as insulin, liver extract, and antibiotic products are not classified as biologics

The broad term “biologics” thus includes the immunizing biologicals which are derivatives of animals (serums,
antitoxins, and globulins) or of microscopic plant organisms (vaccines, toxins, toxoids, and tuberculins), that
either directly or indirectly confer a state of protection against pathogenic microorganisms.

Since these products don’t affect the microorganisms directly, they cannot be considered chemotherapeutic
agents; neither can they be classified with the antibiotics.

Classification of biologics
Biologics can be classified into two general categories, i.e., antigens and antibodies

1. Antigens
• An antigen is a molecule or a molecular structure or any foreign particulate matter or a pollen grain which
induces an immune response in the body, especially the production of antibodies
• An antigen can be defined under three categories, i.e., biological, chemical, and physical
▪ Biologically, it is a substance, which when introduced into the tissues of man or other vertebrates
will cause the formation of antibodies
▪ Antigen possesses following biological properties
o Immunogenicity: The capacity to induce antibody formation
o Specificity: It is governed by small chemical sites on the antigen molecule called the antigenic
determinants
This is the site with which antibody combines
o Considered foreign: The antigen must be considered foreign by the antibody forming host
▪ Chemically, antigens are usually protein; however, some high molecular weight polysaccharides are
antigenic
▪ Physically, antigens must possess high molecular weight, i.e., more than 10,000
o The high molecular weight is associated with immunogenicity
• Examples of antigens
▪ Exotoxins
▪ Proteins and polysaccharides on the cell surface and capsules of bacteria
▪ Protein coat of virus particles
• Microorganisms contain not one but many antigens, which, in turn, may contain many antigenic
determinants
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2. Antibodies
• An antibody is a blood protein, produced in response to and counteracting a specific antigen
• Antibodies are produced by plasma cells (a type of white blood cell)
• Antibodies are found predominantly in the serum fraction of blood
• They also exist in other body fluids and in association with other tissues like lymph nodes and mucous
membrane
• When serum proteins are separated by electrophoresis, four predominant fractions are obtained
i. Serum albumin
ii. Alpha globulin
iii. Beta globulin
iv. Gamma globulin (antibodies occur in this fraction and are called immunoglobulins)

Classification of antibodies (immunoglobulins)

• Classification on the basis of physical, chemical, and immunological properties
i. Immunoglobulin A (IgA)
ii. Immunoglobulin D (IgD)
iii. Immunoglobulin E (IgE)
iv. Immunoglobulin G (IgG)
v. Immunoglobulin M (IgM)

Haptens
• Compounds with a molecular weight lower than 10,000, are partial antigens, called haptens
• Due to low molecular weight, they lack the property of immunogenicity
• However, they can attach to host proteins to form a complete antigen
• Example: Drugs or their breakdown products may act as haptens, e.g., penicillin allergy

Immunity
• Immunity is the state or quality of being resistant to a particular infectious disease or pathogen
• Immunity is the immune system’s way of protecting the body against an infectious disease
• Immunity is classified into 2 major types
Innate (natural) immunity Acquired immunity
• The term natural or innate means the defense mechanisms that are present in the body because of race,
species specificity, and a multitude of other factors not easily defined
▪ It does not include any mechanisms especially developed during the lifetime of the individual
• Natural immunity is endowed at birth and is retained because of an individual’s constitution
• Acquired immunity is a type of immunity that develops when a person’s immune system responds to a
foreign substance or microorganism, or that occurs after a person receives antibodies from another
source
• Acquired immunity is quite specific
• Acquired immunity is generally subdivided into 2 classes
1. Active immunity 2. Passive immunity

1. Active immunity
• Active immunity means the specific immunity developed by an individual in response to the introduction
of antigenic substances into the body
• Active immunity is developed gradually/slowly
• Active immunity is usually long-lasting
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Active immunity can be divided into two categories

A. Naturally acquired active immunity
• In this type of immunity, the antigenic substances are received by the body in a natural manner
• For example, recovery from an infection, such as measles or scarlet fever, produces an immunity
▪ This immunity is acquired naturally

B. Artificially acquired active immunity

• In this type of immunity, the antigenic substances are received by the body through the administration
of a vaccine or toxoid
• This immunity may be produced as the response to a series of injections
▪ Thus, stimulating the body cells to make their own antibodies and producing an immunity
▪ For example, typhoid vaccine produces active immunity
▪ This immunity is acquired artificially

2. Passive immunity
• Passive immunity is the type developed by the introduction of preformed antibodies (not antigens) into
the body
• In this type, the body cells are not stimulated to produce their own antibodies
• This immunity is produced quickly
• Passive immunity is not long-lasting

Passive immunity can be divided into two categories

A. Naturally acquired passive immunity
• The immunity developed in a newborn infant through transmission of the antibodies from the blood of
the mother

B. Artificially acquired passive immunity

• The injection of immunizing biologics containing preformed antibodies in forms produces artificially
acquired passive immunity
• Examples: Mumps immune globulin, antirabies serum, etc.

• In case of active immunity, depending on the nature of the antigen and the site of injection, antibody
can be detected in the serum several days after the first injection of antigen
• The antibody titer rises gradually to a low peak after the first injection (primary response)
▪ The antibody titer rises immediately after subsequent (second, third, etc.) injections
▪ The antibody titer then falls slowly over a period of months
• A second injection of antigen, administered while antibodies from the first stimulus are still present,
results in a rapid rise to a much higher peak than with the first injection (secondary response)
• The second injection should not be too close in time to the first injection
▪ If so, there is no additional effect on antibody production
• The antibodies disappear much more slowly after the second stimulus than after the first
• There is rapid rise of antibody titer following a second administration of the antigen (the booster shot)
▪ It presumably indicates that the antibody-producing cells have been primed by the first contact with
antigen
▪ Therefore, respond more effectively and more quickly when they encounter the antigen a second
time
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▪ So, immune cells recognize the invading substance (an antigen) and produce antibodies specific
against that antigen
▪ This phenomenon is termed as the recall or anamnestic phenomenon (Figure 1)
• The major cellular components of the immune system are the macrophages and the lymphocytes
• Certain lymphocytes, called B cells, produce antibodies
• B cells arise from the bone marrow in humans
• Immunity by B cells (production of antibodies) is called humoral system of immunity because the B cells
circulate in the body fluids, primarily in blood

Figure 1. An illustration of the recall or anamnestic phenomenon in antibody production

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VACCINES
• A vaccine is a preparation containing weakened or dead microbes of the kind that cause a particular
disease
• Vaccines are administered to stimulate the immune system to produce antibodies against that disease
• Vaccines may contain living, attenuated, or killed viruses, killed rickettsiae, or attenuated or killed
bacteria
• Vaccines are available in dried and in liquid form
• Primary active immunity from vaccination develops more slowly than the incubation period of most
infections
▪ Hence, vaccines must be induced prior to exposure to the infectious agent
▪ Therefore, the general action of vaccines should be considered prophylactic
• One exception is the rabies vaccination
▪ The rabies virus has a median incubation period of 35 days in humans
▪ There is usually sufficient time for protective antibodies to develop when the vaccine is administered
after exposure
• Nonliving vaccines provide protection for only a limited time
▪ Therefore, repeated vaccination is required
▪ For example, to maintain protection against typhoid fever, cholera, plague, and typhus
• Active immunization with living agents is generally preferable to immunization with killed vaccines
▪ They have a superior and more long-lived immune response
▪ For example, a single vaccination of measles, rubella, or mumps vaccine is sufficient to produce a
long-lasting immunity
• Use of vaccines is contraindicated under conditions in which the immune response may he depressed
▪ For example, during therapy involving corticosteroids, antineoplastic agents, immunosuppressive
agents, or radiation
▪ In patients with immunoglobulin deficiency
• Active immunization may cause fever, malaise, and soreness at injection sites
• Allergic reactions may also occur
▪ As a result of either from the organism constituting the vaccine, or
▪ from a protein incorporated into the vaccine during manufacture

VIRAL VACCINES
• Viral vaccines for prophylaxis against mumps, rubella, rubeola, smallpox, and yellow fever contain living
viruses
• Inactivated or killed viruses are used in influenza and rabies vaccines
• Preparations containing live attenuated or killed viruses are available for immunization against
poliomyelitis
• The cultivation of viruses poses a problem because they are completely dependent on living cells for their
sustenance
▪ No method of growing viruses in artificial culture media is known
• A number of viruses currently employed in viral vaccines are grown on tissue cultures prepared from
chick embryo, monkey kidney, or human diploid cells

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Smallpox vaccine
• Small pox vaccine is the living virus of vaccinia (cowpox) that has been grown in the skin of a vaccinated
bovine calf
• It is available in dried and in liquid form
• Liquid vaccine should be stored below 0 °C
• Dried vaccine between 2 °C and 8 °C

Source
• Living virus of vaccinia (cowpox), grown in the skin of a vaccinated bovine calf

Historical background
• The pioneer work of Dr. Edward Jenner established that when a mild case of cowpox is developed by a
person, the same person becomes immune to smallpox
• Using this information, he inoculated a young boy with pus from a milkmaid, infected with cowpox
• Two months later, the boy was inoculated with pus from a patient infected with smallpox, but no disease
developed
• Immunity had been established

Preparation
• Belly of calf is washed and shaved
• Epidermis is scarified, so that serum oozes through the cuts
• The “seed virus” is inoculated into the scarifications by hand rubbing
• Workers are protected by rubber surgical gloves
• Calf is maintained in an aseptic stall
• Calf is given food and water during the growth of the virus
• The vesicles that develop, are removed
▪ Thoroughly triturated, and
Either made into a smooth suspension with an aqueous solution of glycerin or sorbitol
Or reduced to a dried pellet
• The animal must be in good health prior to inoculation
• After the virus is harvested, the animal is killed and a necropsy is performed
• If the organs show no effects of disease from other causes, the virus is deemed satisfactory for
manufacture

Uses
• It is used as prophylactic against smallpox
• It develops active immunity which lasts for about 7 years

Dose
• Percutaneous, contents of 1 capillary tube, by multiple puncture method
• Routine immunization against smallpox is no longer recommended
▪ Because of sever adverse reactions
▪ Because the disease has now undergone complete worldwide eradication
• Smallpox vaccination is now indicated only for laboratory workers directly involved with the virus
• Dryvax®

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Rabies vaccine
• Rabies vaccine, also known as human diploid cell rabies vaccine (HDCV), is a sterile lyophilized
preparation of either the whole virion or subvirion rabies virus
• Both vaccines are supplied as 1 mL, single-dose vials of lyophilized vaccine with accompanying diluent

Source
• The whole virion vaccine is prepared from Wistar rabies virus
▪ Grown in cultures of human diploid embryo lung tissue, and
▪ Inactivated with tri-N-butyl phosphate and β-propiolactone
• The subvirion vaccine is prepared from the Pasteur-derived Pitman-Moore virus
▪ Grown on human diploid cell cultures developed in Europe, and
▪ Inactivated with β-propiolactone

Historical background
• Louis Pasteur is associated with rabies
• L. Pasteur was able to “fix” the virus of rabies, by
▪ Passing it from an infected dog to the brain of a rabbit, and
▪ Then from one rabbit to another, until a uniformity was established, that
▪ Resulted in attenuated virulence for humans
• Pasteur used such a “fixed virus” to achieve active immunity
• This treatment is not a curative treatment
▪ It actually causes the immunization of a patient, bitten by a rabid animal, to develop more quickly
(by producing antibodies) than incubation period of the disease
▪ So, growth of rabies virus is inhibited

Uses and dose

• The vaccine is an active immunizing agent
• Recommended primarily for the prevention of rabies in persons, bitten by an animal, supposed or known
to be rabid
• However; the vaccine may be used for pre-exposure immunization for veterinarians or other high-risk
individuals
• The usual pre-exposure dose is 3 injections of 1 mL of reconstituted vaccine
▪ On each of days 0, 7, and 21
• Post-exposure immunization should be started as quickly as possible, after the wound has been inflicted
▪ The usual administration schedule is 5 injections of 1 mL of reconstituted vaccine
▪ On each of days 0, 3, 7, 14, and 28
• Administered intramuscularly (IM)
• Rabies immune globulin should be administered at the time of the first dose of vaccine for additional
protection, particularly in case of a bite from wild animal
• Imovax® rabies vaccine
• Wyeth® rabies vaccine
• Formerly, vaccine was prepared by growing the virus in the brain tissue of rabbit
• Brain tissue of rabbit contains significant amount of myelin
• Myelin causes paralysis
• With the advent of HDCV, problems with paralysis have been greatly reduced
• Rare cases of Guillain-Barre syndrome have been reported

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▪ However, these patients recovered completely from the paralysis

(Guillain-Barre syndrome is a disorder in which your body's immune system attacks your nerves)

Yellow fever vaccine

• Yellow fever vaccine is an attenuated strain of living yellow fever virus, selected for high antigenic activity
and safety
• Should be stored at a temperature preferably below 0 °C but never above 5 °C

Source
• Yellow fever virus, grown in the living embryo of domestic fowl, Gallus domesticus

Preparation
• The virus infected chick embryo pulp is suspended in water and after appropriate aseptic processing, is
distributed in suitable quantities into ampules and dried from the frozen state
• The ampules are filled with dry nitrogen and flame sealed

Historical background
• Yellow fever or “yellow jack” was considered an endemic disease in certain tropical regions, including
Central America
• Work on the Panama Canal was abandoned by the French because of the terrific death toll caused by
yellow fever
• Through numerous volunteers among the American troops, stationed in Cuba (during the Spanish-
American war), the Aedes mosquito was finally proved to be the vector of the disease

Uses
• Active immunizing agent against yellow fever

Dose
• 0.5 mL; subcutaneous
• YF-VAX®
• Stamaril®

Influenza virus vaccine

• Influenza virus vaccine is a sterile aqueous suspension of suitably inactivated influenza virus types A and
B, either individually or combined, or
▪ Virus subunits prepared from the extra-embryonic fluid of influenza virus-infected chick embryo
• The strains of influenza virus used in the preparation of this vaccine are those designated for the
particular season by the Center for Drugs and Biologics of the Federal FDA
• Influenza viruses have a high degree of strain specificity and of genetic instability
▪ These factors require a continual re-evaluation of the components of influenza virus vaccine and
result in periodic infections of epidemic proportions even among immunized persons
• Most available vaccines are bivalent and contain types A and B virus strains
• Should be stored at a temperature between 2 °C and 8 °C

Source
• Inactivated influenza virus; grown in the chick embryo

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Preparation
• The virus growths are collected, concentrated, refined by ultracentrifugation, and inactivated by UV
radiation

Uses
• Active immunizing agent against influenza

Dose
• IM; 0.5 mL
• Annual vaccination is recommended for individuals in high-risk categories e.g., those who are
immunocompromised, those over 65 years of age, etc.
• Fluarix® (GSK)
• Fluogen®

Poliomyelitis vaccine
Polio
• Polio is caused by a human enterovirus called the poliovirus
• The virus is most often spread by the fecal-oral route
• Poliovirus enters through the mouth and multiplies in the intestine
• Infected individuals shed poliovirus into the environment for several weeks, where it can spread rapidly
through a community, especially in areas of poor sanitation
• Poliomyelitis can affect any age, but primarily involves children ages less than 5 years and can cause
paralysis or even death

Poliomyelitis vaccine
• The first polio vaccine to be widely used in humans, known as inactivated poliovirus vaccine (IPV) or Salk
vaccine, was developed in the early 1950s by American physician Jonas Salk
▪ This vaccine contains killed virus and is given by injection
▪ The large-scale use of IPV began in 1954, when it was administered to American schoolchildren
▪ In the following years, the incidence of polio in the United States fell from 18 cases per 100,000
people to fewer than 2 per 100,000
• In the 1960s, a second type of polio vaccine, known as oral poliovirus vaccine (OPV) or Sabin vaccine,
was developed (named for American physician and microbiologist Albert Sabin)
▪ OPV contains live attenuated (weakened) virus and is given orally
• OPV is administered orally and does not require health professionals or sterile needle syringes
• OPV is easy to administer in mass vaccination that is why used in polio campaigns that take place in
Pakistan
• Moreover, for several weeks after vaccination the vaccine virus replicates in the intestine, is excreted
and can be spread to others in close contact
• This means that in areas with poor hygiene and sanitation, immunization with OPV can result in passive
immunization of people who have not been vaccinated
• IPV is an extremely safe vaccine and highly effective in protecting children from polio
• It produces antibodies in the blood against poliovirus
• Unlike OPV, IPV has limited ability to stop the spread of virus in a community
• This is why in the polio-endemic countries such as Pakistan, OPV is the predominant vaccine used in the
fight to eradicate the virus
• IPV requires trained health workers, as well as sterile injection equipment and procedures
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• Combining OPV and IPV provides stronger protection against polio

• IPV strengthens immunity in the blood while OPV strengthens immunity in the gut
• IPV has been introduced into the routine schedule across Pakistan to give children the best protection
against polio
• The current routine immunization schedule recommends one dose of IPV and multiple doses of OPV for
full protection against polio
• Once polio is eradicated, IPV will be the only vaccine available for routine use

Source
• Poliomyelitis virus strains (type 1: Mahoney strain; type 2: MEF-1 strain; type 3: Saukett strain); grown
separately in cultures of Rhesus monkey kidney tissue
• In addition to the 3 types of poliomyelitis virus that have been cultured and identified, other paralysis-
producing strains undoubtedly exist
• Immunization with one type of virus does not offer protection against the other types
▪ Thus, the current vaccine is a trivalent preparation

Preparation
• To prepare Salk vaccine, the virus strains are grown separately in cultures of Rhesus monkey kidney
tissues, bathed by a complex nutrient fluid containing more than 60 ingredients
• After incubation, the virus is harvested by decanting the nutrient fluid that is clarified by filtration
• Then, formaldehyde is added
• Formaldehyde treated viruses are maintained at 36 °C and pH 7, until all viruses are killed
• A series of tests is performed to ascertain that all viruses are inactivated
• Then the formaldehyde is neutralized and a preservative is added
• The 3 types of viruses are then pooled, resultant mixture is the trivalent vaccine
• For preparation of Sabin vaccine, the method of preparation is same as that for Salk polio vaccine
• The difference is that, in this vaccine, virus strains are not killed by treatment with formaldehyde, instead
the viruses are attenuated

Uses and dose

• To develop active immunity against poliomyelitis
• Salk vaccine provides protection against paralytic poliomyelitis through the stimulation of serum
antibodies specific for types 1, 2, and 3 poliovirus
▪ But, does not cause the inhibition of viral growth in the intestine that characterizes the Sabin vaccine
• Sabin oral vaccine should never be administered parenterally
• Orimune®
• Poliovirus vaccine live oral is generally frozen
▪ When stored at a temperature of -10 °C, the expiration date is not later than 1 year, after date of
manufacture or date of issue
▪ It may be thawed and refrozen not more than 10 times, provided that the thawed material is kept
refrigerated and the total cumulative duration of the thaw is not more than 24 hours

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Measles vaccines
• Vaccines containing live attenuated rubeola (measles) and rubella (German measles) viruses

Rubeola (measles) vaccine

• Measles virus vaccine live or rubeola vaccine is prepared from attenuated viruses derived from the
original Edmonston B strain or the Enders strain
• The Enders strain is a modified Edmonston strain, and it is claimed to have a high degree of antigenicity
with a low incidence of adverse reactions
▪ Coadministration of immune globulin may not be necessary with vaccines employing this strain
• The rubeola virus is grown on cultures of chicken embryo tissue
• The vaccines are available in a lyophilized form
• Should be stored at a temperature between 2 °C and 8 °C

Uses and dose

• Rubeola vaccine is recommended for active immunization of children 15 months of age or older
• Use in infants under 15 months of age is not recommended
• Good immunity is obtained with a single subcutaneous injection of not less than 1000 TCID50 (tissue
culture infectious doses) of the reconstituted vaccine
The TCID50 is the quantity of virus estimated to infect 50% of inoculated tissue cultures
• Attenuvax®

Rubella (German measles) vaccine

• Rubella virus vaccine live is prepared from the Wistar Institute RA 27/3 strain grown on human diploid
cell tissue
• Rubella vaccine is recommended for active immunization against German measles for children aged 1 to
puberty and for certain other individuals
• This vaccine should not be administered to pregnant or immediate postpartum women
• Special caution must be exercised if it is given to sexually active females
• Precautions must be taken to eliminate the possibility of pregnancy in women of child-bearing age for at
least 3 months following immunization

Uses and dose

• Immunity is obtained with a single subcutaneous injection of not less than 1000 TCID50 of the
reconstituted vaccine
• Use in infants under 1 year of age is not recommended
• Meruvax II®

Mumps vaccine
• Mumps vaccine is the vaccine containing live attenuated mumps virus

Source
• B-level Jeryl Lynn strain of the virus; grown in the cell culture of chicken embryo

Uses and dose

• To produce active immunity against mumps
• It provides active immunity for at least 10 years after immunization
• It is particularly valuable to susceptible individuals approaching puberty and to adults
• It is not recommended for infants less than 1 year old

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▪ Because they may retain maternal mumps antibodies that may interfere with the immune response
• The vaccine is available in a lyophilized form
• Immunization involves a single subcutaneous injection of not less than 5000 TCID50 of mumps virus
vaccine
• Mumpsvax®

Combination virus vaccines

• Combination live virus vaccines are available
▪ Measles + Rubella; Mumps + Rubella; Measles + Rubella + Mumps
• These combination vaccines are administered subcutaneously to children 15 months of age or older
• Use in infants under 15 months of age is not recommended
• Priorix® (GSK) [Measles + Mumps + Rubella]
• M-M-R II® (MSD) [Measles + Mumps + Rubella]

Hepatitis vaccine
• Hepatitis B vaccine is composed of chemically inactivated hepatitis B surface antigen (HBsAg) particles
• HBsAg particles are obtained from the plasma of healthy chronic HBsAg carriers
• Specific antibody (anti-HBs) develops in 75 to 90% of healthy adults after the first 2 doses of vaccine; and
in 85 to 90% after the third dose
• Vaccine-induced antibody has persisted for at least 3 years
• Booster doses are also required

Uses and dose

• Vaccination is recommended for individuals in high-risk categories
• The vaccine is given intramuscularly (IM)
▪ 3 doses of 1 mL (20 μg)
▪ The first 2 doses one month apart, and
▪ A booster dose, administered 6 months after the first dose
• For patients on dialysis and others who are immunocompromised
▪ 3 doses of 2 mL (40 μg) should be used
• For children under 10 years old
• 3 doses of 0.5 mL (10 μg) are recommended
• Engerix B® (GSK)
• Euvax-B®

RICKETTSIAL VACCINES
• Rickettsial diseases are a group of diseases with similar symptoms caused by bacteria of the genus
Rickettsia
• The rickettsiae are a diverse collection of intracellular Gram-negative bacteria
▪ Found in ticks, lice, fleas, mites, and mammals
• These bacteria are spread by the bite of an infected tick or mite

Typhus vaccine
• Typhus vaccine is a sterile suspension of the killed rickettsial organism Rickettsia prowazekii, selected for
antigenic efficiency
• Should be stored at a temperature between 2°C and 8°C
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Source
• Rickettsia prowazekii; grown in the yolk sac membrane of the developing embryo of Gallus domesticus

Uses
• To develop active immunity against epidemic typhus fever

Dose
• Subcutaneous; 2 injections of 0.5 mL, 4 or more weeks apart
• Followed by 0.5 mL every 6 to 12 months, as long as protection is required

Rocky Mountain spotted fever vaccine

• Rocky Mountain spotted fever vaccine is a sterile suspension of the killed rickettsial organism Rickettsia
rickettsii, selected for antigenic efficiency
• Should be stored at a temperature between 2 °C and 8 °C

Source
• Rickettsia rickettsii; grown in the yolk sac membrane of the developing embryo of Gallus domesticus

Uses
• To develop active immunity against rocky mountain spotted fever

Dose
• Subcutaneous; 3 injections of 1mL, each at 7 to 10 days interval
• An annual 1mL booster injection is highly recommended

BACTERIAL VACCINES
• Bacterial vaccines consist of suspensions of attenuated or, more commonly, killed pathogenic bacteria in
isotonic sodium chloride solution or other suitable diluents
• The strains of bacteria employed in preparation of the vaccines must be selected for high antigenicity
• A measure of the potency of a vaccine may be expressed as the number of organisms per unit volume or
as biologic reference units
• Suspensions of young, living organisms grown in standard culture media are killed
▪ Chemically
▪ By application of moist heat at a temperature slightly above the thermal death point, or
▪ by exposure to ultraviolet light
• The smooth or “S” strains of bacteria are uniformly more antigenic than the rough or “R” strains
• Occasionally, stock cultures lose their antigenic qualities, and care must be exercised in a biologic
manufacturer’s laboratory to ensure the use of suitable strains

Typhoid vaccine / Enteric vaccine

• Typhoid vaccine is a sterile suspension containing killed typhoid bacilli, Salmonella typhi
• It contains approximately 1 billion typhoid organisms in each mL
• Typhoid vaccine has been called enteric vaccine because it prevents the effect of the disease on the
intestinal tract

Source
• Killed typhoid bacilli Salmonella typhi

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Uses
• To produce active immunity against typhoid fever
• It is recommended for persons who have had household contact with a known, typhoid carrier, or
▪ For travelers going to areas of the world where typhoid fever is endemic

Dose
• Subcutaneously
▪ 2 injections of 0.5 mL, 4 weeks apart
▪ Followed by 0.5 mL every 3 years thereafter
• Booster injection is recommended when danger of typhoid occurs
• Typherix® (GSK)

Cholera vaccine
• Cholera vaccine is a sterile suspension containing killed cholera vibrio, Vibrio cholerae

Source
• Equal portions of suspensions of killed cholera vibrios (Vibrio cholerae) of the Inaba and Ogava strains
• It contains approximately 8 billion cholera organisms in each mL

Uses
• To produce active immunity against cholera

Dose
• Oral
▪ 2 doses for adults and children from 6 years of age
▪ Children 2 to below 6 years of age should receive 3 doses
▪ Doses are to be administered at intervals of at least one week
• Dukoral® (Crucell)

Plague vaccine

• Plague vaccine is a sterile suspension containing killed plague bacilli, Yersinia pestis
• Rats serve as an animal reservoir for the organisms, but the disease is transmitted to humans through
the bites of fleas that infest the rats
• With rat control and largescale vaccination, plague can be eliminated

Source
• Killed plague bacilli Yersinia pestis
• It contains approximately 2 billion plague organisms in each mL

Uses
• To produce active immunity against plague
• Its use is generally restricted to travelers to Southeastern Asia and to persons who have frequent contact
with wild rodents

Dose
• IM; 2 injections of 0.5 ml, 4 weeks apart; Then 0.2 ml 4 to 12 week later

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Pertussis vaccine
• Pertussis vaccine is a sterile suspension containing killed pertussis bacilli Bordetella pertussis
• It has a potency of 12 protective units per dose
• Should be stored at a temperature between 2 °C and 8 °C

Source
• Killed pertussis bacilli, Bordetella pertussis

Pertussis
• It is also called whooping cough
• Cough is caused by a toxin in the bacterial body
• The organisms attach themselves to the cilia of epithelial cells in the trachea and the irritation produced
provokes the cough spasm

Uses
• To produce active immunity against pertussis

Dose
• Subcutaneous; 3 injections of 0.5 or 1 ml, 3 to 4 weeks apart

Adsorbed pertussis vaccine

• Pertussis vaccine that has been precipitated or adsorbed by the addition of aluminum hydroxide or
aluminum phosphate and resuspended

Uses and dose

• Same as that of pertussis vaccine
• Route of administration is IM

BCG vaccine
• BCG vaccine is a dried, living attenuated culture of the bacillus Calmette-Guerin (BCG) strain of
Mycobacterium tuberculosis var. bovis
• BCG has been used since 1921 globally

Source
• Bacillus Calmette-Guerin (BCG) strain of Mycobacterium tuberculosis var. bovis

Uses and dose

• To produce active immunity against tuberculosis
• Immunological protection against TB is only relative and is not permanent or predictable
• Vaccine is recommended primarily for use for people whose exposure to TB is high
• It should be used only with individuals who give negative tuberculin skin test
• Infants in Pakistan are vaccinated with BCG vaccine because it can prevent severe tuberculosis in children
• BCG vaccine does not provide 100% protection against TB
▪ It significantly decreases the chances of your baby getting this serious disease
• A single dose of BCG vaccine is administered routinely at birth in Pakistan
• The vaccine is administered just beneath the skin
• If the child misses the dose at birth, it can be administered later on as well

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Pneumococcal vaccine
• Pneumococcal vaccine polyvalent affords protection against the 23 most prevalent capsular types of
pneumococci, which account for at least 90% of pneumococcal disease
• it is prepared by isolating and purifying the polysaccharide antigens from strains of Streptococcus
pneumoniae that contain these serotypes

Uses and dose

• Use of this vaccine is indicated for those 2 years of age or older in whom there is an increased risk of
morbidity and mortality from pneumococcal pneumonia
• Even with current antibiotic therapy, the mortality rate in high-risk patients hospitalized with
pneumococcal infection has remained higher than 25%
• The vaccine is administered as a single 0.5 mL dose given either subcutaneously or intramuscularly
• Severe local reactions have occurred after a second dose
• Therefore, more than one dose is not recommended, even for patients who received an older vaccine
that contained fewer pneumococcal types
• Pneumovax®

Haemophilus vaccine
• Haemophilus b polysaccharide vaccine is composed of the purified, capsular polysaccharide of
Haemophilus influenzae type b (Hib)
• Virtually all cases of Haemophilus influenzae meningitis among children are caused by strains of Hib
• Despite effective antimicrobial therapy, the mortality rate from Haemophilus meningitis ranges from 5
to 10%
▪ About one third of the survivors have some form of permanent injury to the central nervous system
• In addition, Hib can cause epiglottitis, osteomyelitis, arthritis, cellulitis, and pneumonia in children

Uses and dose

• Immunization is recommended for all children when they reach 2 years of age and possibly for children
2 to 5 years old who have not been previously immunized
• Most unimmunized children over 5 years old and most adults have protective titers of naturally acquired
antibodies
• The vaccine is administered subcutaneously as a single 0.5 mL dose
• Hiberix®

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TOXINS, TOXOIDS, and ANTITOXINS

• Toxins are bacterial waste products which are considered poisonous to the animal body
▪ Toxins act as antigens
▪ They stimulate the body to produce antibodies, called antitoxins
• When toxins are excreted from bacterial cells and are dissolved in the surrounding culture, they are
referred to as exotoxins
• Endotoxins are toxic substance bound to the bacterial cell wall and released when the bacterium
ruptures or disintegrates
▪ For example, lipopolysaccharides
• A toxoid is a chemically modified toxin from a pathogenic microorganism, which is no longer toxic
▪ But it is still antigenic and can be used as a vaccine
▪ For example, tetanus toxoid, etc.

Preparation of antitoxins
• Specific bacterial exotoxins are injected to the horse, repeatedly
• Antitoxins, against these injected exotoxins, are produced in the horse
• Blood samples are taken from the horse, tests are performed to check the level of antitoxins
• Horse is bled, clot is permitted to form, clear supernatant serum is separated for processing
• Antitoxins are standardized in terms of antitoxin units

TOXINS

Diagnostic diphtheria toxin (Schick test toxin)

• Diagnostic diphtheria toxin is a sterile solution of the diluted, standardized toxic products of growth
of the diphtheria bacillus Corynebacterium diphtheriae

Dose
• Intradermal, 0.1 mL

Uses
• Used to determine the susceptibility of the patient to diphtheria

TOXOIDS

Tetanus toxoid
• Tetanus is a serious but rare condition that can be fatal if untreated
• The bacteria that can cause tetanus can enter our body through a wound or cut in our skin
▪ These bacteria are often found in soil and manure
• Tetanus toxoid is a sterile suspension of purified toxoid obtained from tetanus bacterium Clostridium
tetani
• The steps of its production are; cultivation of C. tetani bacteria, inactivation (detoxification) of tetanus
toxin, purification of tetanus toxoid, mixing of bulk vaccine, and aseptic filling

Uses and dose

• Tetanus toxoid is used to prevent tetanus
• Dose is 0.5 mL intramuscular injection at any age
• Imatet®
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Emergency prophylaxis
• Emergency prophylaxis in case of wounds / injuries suspected of tetanus contamination, as follows
• If tetanus vaccination or revaccination was performed a year or less before present injury
▪ Prophylaxis is not necessary
• If tetanus vaccination or revaccination was performed one to five years previously
▪ A booster dose of 0.5 ml of Imatet must be given intramuscularly
• If vaccination or revaccination was performed more than five years before
▪ It is necessary to give a booster dose of 0.5 mL tetanus toxoid and also an injection of 1500 IU
tetanus antitoxin or tetanus immunoglobulin
• If vaccination or revaccination was not carried out or was incomplete
▪ Tetanus antiserum as recommended in above and a complete course of tetanus vaccination must
be performed

ANTITOXINS

Diphtheria antitoxin
• Diphtheria antitoxin is a sterile, non-pyrogenic solution of the refined and concentrated proteins,
chiefly globulins, containing antitoxic antibodies obtained from the blood serum or plasma of healthy
horse, that has been immunized against diphtheria toxin or toxoid
• It has a potency of not less than 500 antitoxin units per mL
• Should be stored at a temperature between 2 °C and 8 °C

Source
• Antitoxic antibodies obtained from the blood serum or plasma of healthy horse, that has been
immunized against diphtheria toxin or toxoid

Uses
• To produce passive immunity against diphtheria
• Penicillin and other antibiotics kill the diphtheria organisms, but they have no effect on the toxins
• Any person with clinical symptoms of diphtheria should receive the antitoxin at once without waiting
for bacteriologic confirmation

Dose
• IM or IV
• Prophylactic: 1000 to 10,000 units
• Therapeutic: 20,000 to 120,000 units

Tetanus antitoxin
• Tetanus antitoxin is a sterile, non-pyrogenic solution of the refined and concentrated proteins, chiefly
globulins, containing antitoxic antibodies obtained from the blood serum or plasma of healthy horse,
that has been immunized against tetanus toxin or toxoid
• It has a potency of not less than 400 antitoxin units per mL
• Should be stored at a temperature between 2 °C and 8 °C

Source
• Antitoxic antibodies obtained from the blood serum or plasma of healthy horse, that has been
immunized against tetanus toxin or toxoid

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Uses
• Tetanus antitoxin is employed in the treatment and prophylaxis of tetanus, if tetanus immune globulin
is not available
• It creates passive immunity to tetanus
• Like diphtheria antitoxin, it is a valuable therapeutic agent when used early in the disease
• Prophylactic doses should be given to individuals who have had 2 or less injections of tetanus toxoid
and who have tetanus-prone injuries that are more than 24 hours old
• Tetanus toxoid should also be administered at a different site on the patient

Dose
• Subcutaneous or IM
• Prophylactic
▪ 1500 to 5000 units
• Therapeutic
▪ 50,000 to 100,000 units or more, with at least part of the dose given intravenously

Vaccination schedule for Pakistan

BCG: Bacillus Calmette-Guerin, tuberculosis vaccine

IPV: Inactivated poliovirus vaccine
MR: Measles, mumps, and rubella
OPV: Oral poliovirus vaccine

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BIOLOGICS

VENOMS and ANTIVENINS

• Venom is a poisonous substance secreted by animals such as snakes, spiders, and scorpions and
typically injected into prey or aggressors by biting or stinging
• Can be compared with exotoxins of bacteria
• Antivenin (antivenom) is an antiserum containing antibodies against specific poisons, especially those
in the venom of snakes, spiders, and scorpions
▪ Example: Anti-snake venom serum (polyvalent equine immunoglobulins)

Preparation of antivenins
• Antivenins are prepared by injecting the specific venoms to the horses, in a manner similar to that for
antitoxins

Anti-snake venom serum (Polyvalent equine immunoglobulins)

• Anti-Snake venom serum is a sterile preparation containing purified and concentrated
immunoglobulins obtained from the serum of healthy horses immunized against the venoms of the
following four common poisonous snakes of Pakistan
Cobra (Naja naja)
Krait (Bungarus caeruleus)
Russell's viper (Vipera russelli)
Saw scaled viper (Echis carinatus)

Dosage
• Conventionally the dose of anti-snake venom serum is 10-30 mL
▪ In severe cases it may go up to 200 ml
• One third of the initial dose can be administered locally around the wound
▪ Remaining two third of the dose intravenously
• The second dose can be repeated two hours after the first dose or even earlier depending on the
condition of the patient and severity of symptoms
• Further doses can be repeated after six hours interval until the symptoms disappear completely
• The dosage, schedule of repeat dosages, and route of administration of anti-snake venom serum may
be modified or adjusted by the specialist/practitioner according to the severity of symptoms

Manufactured By:
• Biological Production Division, National Institute of Health, Islamabad, Pakistan

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ANTISERUMS
Antiserums
• Antiserum (plural: antisera) is blood serum containing polyclonal antibodies and is used to pass on
passive immunity to many diseases
• Examples: Anti-rabies serum, etc.

Preparation of antiserum
• Antiserums are prepared by injecting the specific bacteria or viruses to the horses, in a manner similar
to that for antitoxins
• The difference is that, here bacteria or viruses (not toxins) are used for production of specific
antibodies

Human immune serum globulins (immune globulins)

• A sterile solution of globulins derived from pooled human blood that contains antibodies that are
normally present in the blood of adults, used as a passive immunizing agent
• Individuals are hyperimmunized against a particular disease
▪ Antibodies formed in these individuals are removed
• These are better than antiserums, as chances of sensitization are less
• Examples: Mumps immune globulin, Rho (D) immune human globulin, etc.

Antirabies serum
• Antirabies serum is a non-pyrogenic solution containing antiviral substances obtained from the blood
serum or plasma of a healthy horse that has been immunized against rabies by means of vaccine
• New concept in immunization is, administration of antirabies serum in conjunction with rabies vaccine
• Now a days, rabies immune globulin is preferred

Uses
• Immediate protection against rabies

Dose
• IM; 70 units per Kg body weight

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PLANT-BASED BIOLOGICS
• During the last century, the trend upturned and researchers began focusing on biological
manufacturing
• Traditional techniques to produce biologics use microbial fermentation or animal cell culture (as
discussed in this chapter)
• New concept is plant-based biologics
▪ Plant-based technologies involve the integration of the desired genes, encoding the antigen
protein for specific disease, into the genome of plant tissues by various methods
▪ This technology will provide cost-effective biologics with easy production and immediate access
▪ This technology is foreseen to expand its uses in the upcoming years

Reading References
• Tyler VE, Brady LR, Robbers JE. Pharmacognosy. Lea & Febiger, 7th edition, 1976
• Tyler VE, Brady LR, Robbers JE. Pharmacognosy. Lea & Febiger, 9th edition, 2003
• Laere et al. Plant-based vaccines: production and challenges. Journal of Botany, 2016. DOI:
10.1155/2016/4928637
• Chen et al. Plant-made biologics. Biomed Res Int., 2014. DOI: 10.1155/2014/418064
• Plant-based biologics: an Effective way to combat life-threatening diseases (www.researchdive.com)
• www.amson.org.pk
• www.nih.org.pk
• www.endpolio.com.pk
• www.druginfosys.com
• www.epi.gov.pk

PharmD Session: 2022-2023

To notify mistakes and for suggestions, kindly contact

Hafiz Abdul Khaliq ([email protected])

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