Contents:
Introduction
Polio (or acute anterior poliomyelitis) — is an acute infections inflammation of the anterior horns of the gray matter of the spinal cord. In this acute systemic infections disease, paralysis may or may not occur. In the majority of patients, the disease is mild, being limited to respiratory and gastrointestinal symptoms, such constituting the minor illness or the abortive type, which lasts only a few days. In the major illness, muscle paralysis or weakness takes place with loss of superficial and deep reflexes. In such instances characteristic lesions are found in the gray matter of the spinal cord, medulla, motor area of the cerebral cortex and the cerebellum. It has been observed that probably 90% of infections caused no symptoms and only 1% lead to actual paralysis.
Explanation. The causation of the disease is due to the fact that the virus first invades the cell of the oropharyngeal and the intestinal mucosal ; and in most individual remains here until it gets eliminated by cell replacement completely. In a rather small percentage of infections, however it finds its way via the lymphatics route and the blood cell to the CNS where it produces the ensuing ‘degenerative changes’ which ultimately is responsible for causing paralysis. The first successful prophylactic was due to the inactivated vaccine which is administered parenterally and thereby stimulates the production of antibodies in the blood. As the virus pass through the blood stream on its way from the alimentary canal to the CNS ; it gets neutralized subsequently thereby providing the protection. However, it fails to prevent establishment of the infective virus in the mucosa. Interestingly, the individual actively immunized with this type of vaccine might act as a ‘carrier for virulent virus’ and hence infect others.
The second successful type of vaccine contains ‘attenuated organisms’ which on being administered to an individual orally (e.g., on a sugar lump) invariably invade by the normal route ; and in the mucosa of the alimentary canal to check and pervent the establishment of the infective virus by stimulating the production of antibodies locally and possibly through the interporal production. In this case also the antibodies develop in the blood stream.
In short, both these types of vaccines are duly represented in the Indian Pharmacopoea (IP) ; however, in actual practice the ‘Live Oral Vaccine’ is largely preferred.
Variants of Polio Vaccine : There are in all five recognized variants of the ‘polio vaccine’, namely :
(a) Oral Polio Vaccine (OPV),
(b) Salk Type Polio Vaccine,
(c) Sabin Type Polio Vaccine,
(d) Inactivated Poliomylitis Vaccine [or Inactivated Polio Vaccine BP-1993], and
(e) Poliomyelitis Vaccine Live (Oral) [or Polio Vaccine Live (Oral) BP-1993].
These different variants of ‘Polio Vaccine’ shall now be treated individually in the sections that follows :
Oral Polio Vaccine (OPV)
It was intially developed by Sabin, after the American Researcher, and is a mixture of ‘live attenuated strains’ of the three polio viruses viz., Type 1, 2, and 3.
- An oral dose of the mixture is given to the children at 3 months age ; 4to 5 months ; and finally at 8-11 months of age. These doses are usually administered at the same time as and when the initial course of injections of triple DTP vaccination are given to a child.
- A booster dose is administered orally at 4 to 5 and again at 15-18 years of age.
- The viruses invariably grow in the ‘lymphoid tissue’ associated with the gut epithelium and thus generate local and humoral immunity.
- As fecal excretion of the vaccine strains may actually persist for some weeks, the nearest family members of the children given OPV must be advised with respect to the ‘hygenic handling and proper disposal of napkins / diapers etc.,’ Any non-immune immediate family members should preferably be immunized simultaneously.
- Vaccine associated poliomyelitis takes place very rarely (less than one in a million) ; however, the benefits of the vaccine outweighs this enormously.
- OPV must not be administered to hypogammaglobulinaemic children specifically.
- Absorption of antigen or preformed antibodies via the alimentary canal has not been of much interest in the therapeutic armamentarium until recently because of the major reason that the ‘prevailing digestive process’ invariably gives rise to severe wastage.
Salk Type Polio Vaccine
The Salk Type Polio Vaccine (or Salk filled vaccine) is a formaldehyde inactivated mixture of the three types of polio virus and it is also found to be very effective. It was subsequently replaced more or less by the ‘Sabin type polio vaccine’ in a good number of countries based on the following predominant plus points, namely :
- If refrigeration is assured adequately, OPV may be more conveniently delivered to communities ; and that too at a reasonably, economically viable and cheaper cost.
- Local gut immunity associated with OPV coupled with the possibility of the vaccines’ spread and separation of wild virus in the community is an additional advantage. The Salk type polio vaccine is, in fact, an inactivated vaccine baptized after the American ‘virologist’ who first developed it.
Preparation. The various stages involved in its preparation are as follows :
(1) The three types of polio vaccines (e.g., Type 1, 2 and 3) are grown individually in either suspended or fixed cell cultures of monkey kidney tissue ; and for this Rhesus monkeys are employed generally.
(2) Rhesus monkeys are usually quarantined on arrival and checked meticulously for TB and other ‘communicable diseases’ both before and after death.
(3) The ‘monkey kidney cell’ must not have been propogated in series and are invariably obtained from a continuous line of cell. This exclusion, however, is entirely based on the possible fear that because it has been easier to ‘produce continuous lines of malignant cell’ in comparison to the ‘normal cell’ ; all this vividly depict the ability to maintain a line of the latter which is indicative of a transformation towards malignancy in the cells.
(4) For both types of vaccines, the inclusion of serum is strictly forbidden in the culture media employed for maintaining cell growth in the course of ‘virus propogation’ ; however, it may be included in media used to initiate the process of growth of tissue cells. It is so done in order to prevent the ‘serum reactions’ when such preparations are subsequently administered. In fact, it is virtually more important in the inactivated vaccine which is usually given parenterally ; and for this purpose there is a prescribed limit of 1 ppm of serum in the final product.
(5) Healthy Rhesus monkeys are duly anaesthetized and their kidneys are removed and decapsulated. The ‘cordical tissue’ is coarsely disintegrated and suspended in No : 199 substrated.
(6) The chopped tissue is then treated with several lots of dilute, warm trypsin solution each at 0.5% concentration at pH 7.6 for a span of 20 minutes. This treatment, in fact, partially hydrolyses the tissue frame-work ; and further helps in separating the cells into a rather free suspension without affecting their viability and efficacy.
(7) The cells are subsequently centrifuged, washed and resuspended in a complex medium to a density of 3 × 105 mL– 1. The medium could be either an admixture of No : 199 substrate plus calf-serum or an admixture of lactoalbumin hydrolysate plus serum.
(8) The resulting suspension is then inoculated into relatively larger vessels and incubated for a duration of 5 days to allow the cells to become adequately established as a ‘monolayer’ on the glass surface.
(9) When optimal growth has taken place, the liquior is poured off and the cells are washed with BSS-medium. After adding fresh medium, a small inoculation with one specific strain on the ‘virulent stock’ virus is made.
(10) Separate batches for each of the three types are made and all are incubated for approximately three days or until such a time the full effect of the virus degeneration has actually taken place. By this time the medium invariably contains a high concentration of the ‘free virus particles’.
(11) Debris is centrifuged off and the supernatant layer is cooled adequately. In case, these are not required immediately, the deep frozen strains may still be kept separately which can now be batches to larger volume(s) as and when required.
(12) Once this harvesting step is over, the resulting virus suspension is tested meticulously to confirm that only the correct strain of polio virus is present ; and also that the virus liter is above certain specified bare minimum level and in addition free from viral, bacterial and fungal contaminants as far as possible.
(13) Consequently, the suspension is made to pass through the filters having increasing fineness so as to remove any remmants of tissue cells and ultimately ‘bacteria’. The former could some of the virus from the inactivating agent.
(14) The liquors containing the virus particles are separately treated with dilute (0.01% v/v) formaldehyde under accurately controlled conditions of pH and temperature and making use of a magnetic stirrer. However, the removal of tissue cells and finally bacteria is duly accomplished within a span of 6 days, but in actual practice at least twice this duration is allowed (i.e., 12 days) to ascertain almost 100% absence of any ‘active virus’.
(15) In usual practice, the suspension may be refiltered at the half-way stage i.e., after nearly 7 days. The rate at which the phenomenon of inactivation takes its normal course is followed meticulously for several days at a stretch on regular intervals ; and subsequently almost between the 9th and 12th days larger samples are tested precisely for the total and absolute absence the infecting virus.
(16) The formalized and sterile viral solution is now subjected to dialysis and checked thoroughly. The ‘univalent vaccines’ of the three strains are now blended adequately to give rise to the desired ‘trivalent product’. At this critical stage large number samples are rechecked once again for freedom from the infective virus ; and finally the added formaldehyde solution is carefully neutralized with the addition of sodium metabisulphite.
(17) A requisite quantum of ‘thimerosal’ is added to serve as a bactericide. An aliquot of soluble disodium edetate is also included with a specific purpose to sequester heavy metals (as ‘chelates’) that would catalyze the decomposition of thiomersal to such products which are ‘toxic’ to the virus.
(18) The entire sequential procedure right from the very beginning to the final stage usually takes about 120 days. Importantly, the ‘toxoid’ is prepared for IM-injection by subjected it to due emulsification with the aid of mineral oil essentially containing 3% highly purified mannite monoleate in almost equal proportions together with 0.01% thimerosal as preservative.
Sabin Type Polio Vaccine
Interestingly, the vigorous activity in this direction has revived once again with the advent of enormous development and broad-scale usage of oral polio vaccine (OPV), that essentially comprises of living attenuated strains of the virus. However, the principle of its action is that these virus particles have the ability to proliferate in the gut and consequently release their modified toxins that are in turn get absorbed directly into the blood stream ; and thus induce the formation of specific antibodies.
Preparation. The various steps involved are as follows :
(1) The overall manufacturing procedure is essentially as that of the ‘Salk Vaccine’ except in one aspect that once the attenuated strains prepared by rapid passages through tissue cultures of monkey kidney cells are employed exclusively.
(2) The virus in the ‘final vaccine’ must not represent more than three sub-cultures from a strain that laboratory and clinical test have shown to be satisfactory. This, however, drastically reduces the chance of using a vaccine which has been rendered either more virulent or lost antigenicity.
(3) Here exists practically no ‘activation stage’ in this specific vaccine.
(4) Besides, testing for freedom on the extraneous bacteria, molds, and viruses, special tests are absolutely predominantly necessary because the virus in the vaccine is living so as to confirm as well as ascertain the absence of virulent polio virus.
Inactivated Poliomyelitis Vaccine [or Inactivated Polio Vaccine BP-1993]
The Inactivated Poliomyelitis Vaccine is an aqueous suspension of appropriate strains of poliomyelitis virus, types 1, 2, and 3, grown in suitable cell cultures and inactivated by a suitale method. It is invariably obtained as a ‘clean liquid’.
Preparation. The various steps followed sequentially are as described under :
(1) It is solely based on a ‘seed-lot system’. The virus used in the final vaccine represents not more than ten subcultures from the seed lots used for the production of the vaccine on which were carried out the laboratory and clinical tests that showed the strains to be suitable.
(2) Animal serum may be employed in the medium for the initial cell growth but the medium for maintaining cell culture during virus multiplication contains no protein. The concentration of serum carried over into the vaccine does not exceed one part per million.
(3) The million may contain a suitable pH indicator, such as : phenol red, and suitable antibodies at the smallest effective concentrations.
(4) Each virus suspension is tested for identity, for bacterial sterility and, after neutralization with ‘specific antiserum’, for affording freedom from extraneous viruses.
(5) The virus suspension is passed through a suitable filter and may then be concentrated and purified.
(6) The suspension should contain at least 7.0 log10 CCID 50 mL– 1 for each type of virus.
(7) Within a suitable period of time of the last filteration, preferably within 24 hours, appropriate chemical substances that inactivate the ‘virus filtrate’ without destroying its antigenicity are added. During the process of inactivation a suitable filtration is carried out. If necessary, the inactivating substance is later neutralized.
(8) Each of the monovalent suspensions is shown by appropriate tests in cell cultures to be free from infective poliomyelitis virus and other human and simian (i.e., monkey like) viruses.
(9) The ‘trivalent vaccine’ is prepared by mixing suspensions of each type.
(10) Before the addition of any antimicrobial preservative, the ‘trivalent suspension’ is shown to be free from infective poliomyletis virus and other human and simian viruses.
Poliomyelitis Vaccine, Live (Oral) [or Polio Vaccine Live (Oral) BP-1993]
Poliomyletis Vaccine, Live (Oral) is an aqueous suspension of suitable live, attenuated strains of poliomyletis virus, types 1, 2 or 3, grown in suitable, approved cell cultures. It may contain any one of the three virus types or mixture of two or three of them. It is a clear a ‘clear liquid’. The vaccine should be shown to be stable.
Preparation. The various steps involved are as enumerated under :
(1) It is based on a seed-lot system. The ‘final vaccine’ represents not more than three subcultures from the vaccine on which were made the laboratory and clinical tests that showed the strains to be suitable as approved by the appropriate authority.
(2) The virus of each type is grown in cultures that have been shown not to contain extraneous microorganisms.
(3) Animal serum may be used in the medium for initial cell growth but the medium for maintaining the cell cultures during virus multiplication contains absolutely no protein.
(4) The cell culture medium may contain a suitable pH indicator e.g., phenol red, and also appropriate antibodies at the smallest effecitve concentrations.
(5) The virus suspension is harvested and is adequately tested for identity, bacterial sterility and freedom from extraneous viruses.
(6) Virus harvests that pass these tests are pooled and filtered through a bacteria retentive filter.
(7) The filtered virus harvest is tested in cell cultures for identity, for growth capacity at different temperatures and for virus concentration.
(8) A test for neurovirulence is carried out by intraspinal injection into Macaca irus (cynomolgus monkey) or equally succeptible animals. The vaccine and a reference homotypic vaccine are examined simultaneously in monkeys from the same quarantine batch.