As with the previous post (029), diphtheria toxoid—that is, detoxified toxin that can still induce an immune response—was the subject of interest for a vaccine against diphtheria. In this study, the researchers were attempting to determine the best dosing schedule and quantity for children ages 1-6.
After immunizing 572 children in total, some with two doses of 0.5 milliliters a month apart and others with 0.5 mL one month and 1 mL the next, they used something called the Schick test to determine whether the children had an adequate immune response from the vaccine. This test involved injecting a small amount of diphtheria toxin under the skin. If the person had antibodies against it, they would neutralize the toxin and show no response; otherwise, they would temporarily have a swollen red spot on their arm. So that meant, a positive test meant non-immune, a negative test meant immune. How accurate this test was, I don't know, but it's better than nothing I guess.
In terms of side effects, the authors didn't observe any problems except for 10 sore arms or general reactions; nothing severe.
Of the children that actually returned after their second dose, the authors found that about 46% of those that got two 0.5-mL doses were Schick-negative (that is, possibly immune) after 4 months. More became Schick-negative over time, so possibly up to 68% overall might be immunized by this dosing.
For those who got one 0.5-mL and one 1-mL, 71% were Schick-negative after 4 months, though many Schick-positive children had been injected from the same vial, so it might've been they just had a bad batch. Excluding those children, about 77% were Schick-negative after 4 months, and 91% after 8 months. So that's better.
Some who were Schick-positive a while after receiving their two doses were given another dose, and some up to four. Those who got three doses were Schick-negative 94% of the time, and all of those that got four doses were eventually Schick-negative (immune).
The authors admit that the sample sizes in this study were too small for definite conclusions, but from what they saw, it seemed like they could give larger quantities for better effect, that doing a Schick test to confirm immunity, and for those that lacked it, another dose should be given. It was important to have a consistent immune response, otherwise people might be overconfident in their protection from the disease, and if vaccinated people kept getting the disease, people might lose confidence in the vaccine.
Next up: Something different.
Citation: Bloomberg, M. W. & Fleming, A. G. Diphtheria Immunization with Diphtheria Toxoid (Anatoxin-Ramon). Can Med Assoc J 17, 801–803 (1927).
Investigating the research on vaccines, one study at a time.
Saturday, October 26, 2013
Saturday, October 19, 2013
029 - The Preparation and Testing of Diphtheria Toxoid (Anatoxine-Ramon)
Diphtheria is an infection with Corynebacterium diphtheriae, which is harmful almost entirely because of the toxin the bacteria produce, called diphtheria toxin. This means that a vaccine need only induce an immune response against the toxin in order to prevent the disease, not against the bacteria themselves.
Before any vaccine was developed, people used something called antitoxin to treat serious diphtheria cases. Antitoxin was made by inoculating an animal with diphtheria toxin and extracting the antibodies that animal produced, then using the antibodies to treat the disease in people. Its effectiveness was somewhat questionable, depending somewhat on whether the illness was caught and treated early enough, but it was the best people had at the time. Actually, the Iditarod dogsled race in Alaska is in memory of a dog named Balto leading a dogsled loaded with antitoxin to Nome, Alaska to treat a serious outbreak there.
Some of the first attempts at vaccine were combining diphtheria toxin with antitoxin and injecting them both into people; the antitoxin would prevent the toxin from causing problems, but the toxin would still be present to induce an immune response against further exposure. This was risky though, since the actual toxin was present and could cause problems if the ratio was wrong; and antitoxin could cause sensitivity problems of its own in some people.
All this makes the current study exciting, as well as the fact that it's the first example of a vaccine in this blog that's close to a kind that's still in use today. It's called a toxoid vaccine. Gaston Ramon was the first to develop it, though he called it anatoxine (being French and all). A toxoid is a toxin that has been detoxified chemically, so it doesn't cause disease, but can still induce an immune response that works against both toxoid and original whole toxin. This makes an ideal vaccine: simple, non-toxic, effective.
The current study describes methods of preparing, testing, and dosing a diphtheria toxoid vaccine. It's not too hard to make, though it takes a while. First, grow the bacteria up in broth, then filter-sterilize the broth (removing all the bacteria), leaving only a solution of toxin. The authors tested the amount of toxin by determining how much of a dose it took to kill half of a number of test animals (the median lethal dose/MLD, also called LD50). Too little toxin would make it ineffective.
Then, to inactivate the toxin, they added a certain amount of formaldehyde, not too much or too little, and heated to body temperature for 1-4 weeks. A pretty long time. They knew it was done by testing it weekly in guinea pigs, injecting a little into the skin and seeing how much skin reaction they got. Once the reaction was smaller than a certain diameter, it was ready. Then they tested it again in guinea pigs for overall toxicity, and for potency (how much immune response it induced in the pigs, measured by protection against whole toxin).
The exact dosing for humans had not been fully determined. They recommended two 0.5mL injections, a month apart, for young adults. It worked better for some people than others, especially well for those that exhibited a skin reaction. Children less than 8 years old rarely had such a reaction; over 8 years, about 25% had a reaction. Older subjects seemed more likely to have a more serious general reaction (headache, fever), but whether this was likely could be tested by injecting a little bit of diluted toxoid right under the skin and seeing if that induced any local reaction. But rarely did anyone have a very serious reaction lasting more than a couple days.
So that's cool, not a clinical trial or anything, but the authors claim they were doing another study of about 50,000 children in Canada, so hopefully I will report on that soon.
Citation: Moloney, P. J. The Preparation and Testing of Diphtheria Toxoid (Anatoxine-Ramon). Am J Public Health (N Y) 16, 1208–1210 (1926).
Before any vaccine was developed, people used something called antitoxin to treat serious diphtheria cases. Antitoxin was made by inoculating an animal with diphtheria toxin and extracting the antibodies that animal produced, then using the antibodies to treat the disease in people. Its effectiveness was somewhat questionable, depending somewhat on whether the illness was caught and treated early enough, but it was the best people had at the time. Actually, the Iditarod dogsled race in Alaska is in memory of a dog named Balto leading a dogsled loaded with antitoxin to Nome, Alaska to treat a serious outbreak there.
Some of the first attempts at vaccine were combining diphtheria toxin with antitoxin and injecting them both into people; the antitoxin would prevent the toxin from causing problems, but the toxin would still be present to induce an immune response against further exposure. This was risky though, since the actual toxin was present and could cause problems if the ratio was wrong; and antitoxin could cause sensitivity problems of its own in some people.
All this makes the current study exciting, as well as the fact that it's the first example of a vaccine in this blog that's close to a kind that's still in use today. It's called a toxoid vaccine. Gaston Ramon was the first to develop it, though he called it anatoxine (being French and all). A toxoid is a toxin that has been detoxified chemically, so it doesn't cause disease, but can still induce an immune response that works against both toxoid and original whole toxin. This makes an ideal vaccine: simple, non-toxic, effective.
The current study describes methods of preparing, testing, and dosing a diphtheria toxoid vaccine. It's not too hard to make, though it takes a while. First, grow the bacteria up in broth, then filter-sterilize the broth (removing all the bacteria), leaving only a solution of toxin. The authors tested the amount of toxin by determining how much of a dose it took to kill half of a number of test animals (the median lethal dose/MLD, also called LD50). Too little toxin would make it ineffective.
Then, to inactivate the toxin, they added a certain amount of formaldehyde, not too much or too little, and heated to body temperature for 1-4 weeks. A pretty long time. They knew it was done by testing it weekly in guinea pigs, injecting a little into the skin and seeing how much skin reaction they got. Once the reaction was smaller than a certain diameter, it was ready. Then they tested it again in guinea pigs for overall toxicity, and for potency (how much immune response it induced in the pigs, measured by protection against whole toxin).
The exact dosing for humans had not been fully determined. They recommended two 0.5mL injections, a month apart, for young adults. It worked better for some people than others, especially well for those that exhibited a skin reaction. Children less than 8 years old rarely had such a reaction; over 8 years, about 25% had a reaction. Older subjects seemed more likely to have a more serious general reaction (headache, fever), but whether this was likely could be tested by injecting a little bit of diluted toxoid right under the skin and seeing if that induced any local reaction. But rarely did anyone have a very serious reaction lasting more than a couple days.
So that's cool, not a clinical trial or anything, but the authors claim they were doing another study of about 50,000 children in Canada, so hopefully I will report on that soon.
Citation: Moloney, P. J. The Preparation and Testing of Diphtheria Toxoid (Anatoxine-Ramon). Am J Public Health (N Y) 16, 1208–1210 (1926).
Tuesday, October 8, 2013
028 - Studies on Pneumococcus Immunity III. the Nature of Pneumococcus Antigen
Well, I've already discussed pneumococcal vaccines a decent amount (017, 018, 019, 021, 023, 024, 026), but none of the vaccines in these studies really resemble the ones used against pneumococcus today, which, instead of using whole killed bacterial cells, use only a component (polysaccharide) of the bacterial capsule as an antigen. So the study today is one done in the early 1920s where people began to discover that it was not necessary or helpful to use whole cells in vaccines.
The purpose was to investigate the chemical nature of the antigen that provided the main protection against infection with pneumococcus bacteria. If people could purify and use just the important component, the vaccines' toxicity would be reduced and the effectiveness increased with lower doses.
So the authors studied the effectiveness of vaccines made by different components of pneumococcus cells, testing their ability to protect white mice against live pneumococcus infection. Unvaccinated control mice all died of infection within 3 days, so test mice were considered "survivors" if they lasted at least 5 days.
Whole-cell killed vaccines protected most mice against infection. When the researchers used different methods to isolate proteins (and "nucleoproteins" containing phosphorus, adenine, and guanine; in other words, nucleic acids) and whatever was attached to them, and made vaccines out of those, they worked decently well also. So it must be protein (or nucleic acid) or something attached to it that induced the immunity.
Following the lead of other researchers, they tried digesting the proteins with proteolytic enzymes, and then seeing if the resulting solutions still worked as vaccines. Depending on the method used, these vaccines were decently effective, especially when the protein was degraded more thoroughly.
Finally, they separated the components in a degraded protein solution that could dissolve in alcohol from those that came out of solution. The latter were very bad as vaccines, but the former were just about as good as whole cells. It protected 2 out of 3 mice against a dose of pathogens that was several million times larger than the minimum lethal dose. So they had gotten as far as they could in isolating the important antigen component, and as far as they could tell, it contained very little protein.
They did some further tests, determining that this antigen could survive well in heated acid but not heated base, and that it could last at least several months when refrigerated, even after being heated pretty hot for an hour. And while the initial experiments were all done with pneumococcus type I, the same type of antigen was present in types II and III. And it was not toxic to mice, as far as they could tell.
So that's pretty interesting as a step forward in pneumococcus vaccine development. The numbers of animals were pretty small for each experiment, and it was done in mice, not humans, but it's important as preliminary data.
The papers that cite this study generally agree that the antigen these authors probably isolated was polysaccharide in nature. As Felton et al. say:
Citation: Perlzweig, W. A. & Steffen, G. I. Studies on Pneumococcus Immunity III. the Nature of Pneumococcus Antigen. J Exp Med 38, 163–182 (1923).
The purpose was to investigate the chemical nature of the antigen that provided the main protection against infection with pneumococcus bacteria. If people could purify and use just the important component, the vaccines' toxicity would be reduced and the effectiveness increased with lower doses.
So the authors studied the effectiveness of vaccines made by different components of pneumococcus cells, testing their ability to protect white mice against live pneumococcus infection. Unvaccinated control mice all died of infection within 3 days, so test mice were considered "survivors" if they lasted at least 5 days.
Whole-cell killed vaccines protected most mice against infection. When the researchers used different methods to isolate proteins (and "nucleoproteins" containing phosphorus, adenine, and guanine; in other words, nucleic acids) and whatever was attached to them, and made vaccines out of those, they worked decently well also. So it must be protein (or nucleic acid) or something attached to it that induced the immunity.
Following the lead of other researchers, they tried digesting the proteins with proteolytic enzymes, and then seeing if the resulting solutions still worked as vaccines. Depending on the method used, these vaccines were decently effective, especially when the protein was degraded more thoroughly.
Finally, they separated the components in a degraded protein solution that could dissolve in alcohol from those that came out of solution. The latter were very bad as vaccines, but the former were just about as good as whole cells. It protected 2 out of 3 mice against a dose of pathogens that was several million times larger than the minimum lethal dose. So they had gotten as far as they could in isolating the important antigen component, and as far as they could tell, it contained very little protein.
They did some further tests, determining that this antigen could survive well in heated acid but not heated base, and that it could last at least several months when refrigerated, even after being heated pretty hot for an hour. And while the initial experiments were all done with pneumococcus type I, the same type of antigen was present in types II and III. And it was not toxic to mice, as far as they could tell.
So that's pretty interesting as a step forward in pneumococcus vaccine development. The numbers of animals were pretty small for each experiment, and it was done in mice, not humans, but it's important as preliminary data.
The papers that cite this study generally agree that the antigen these authors probably isolated was polysaccharide in nature. As Felton et al. say:
"Although not then identified, from the description of their preparation the product undoubtedly contained the capsular polysaccharide."
Citation: Perlzweig, W. A. & Steffen, G. I. Studies on Pneumococcus Immunity III. the Nature of Pneumococcus Antigen. J Exp Med 38, 163–182 (1923).
Sunday, October 6, 2013
027 - Experimental Studies of the Nasopharyngeal Secretions from Influenza Patients X. the Immunizing Effects in Rabbits of Subcutaneous Injections of Killed Cultures of Bacterium pneumosintes
This study,1 and those accompanying it (parts I-IX) seemed like a particularly exciting step forward in the understanding of influenza. Published about 4 years after the worst known epidemic of flu in history (1918 Spanish flu), people were understandably keen on discovering the cause and ways to prevent this disease.
Previous research had suggested bacterial pathogens as the culprit, such as Pfeiffer's bacillus (aka Bacillus influenzae, later named Haemophilus influenzae) or others, but these could not be reliably found in flu patients, nor did serum from patients always contain antibodies against the bacteria. Vaccines made from them (such as in 019 or 026) didn't reliably protect against the flu, though they may have helped with secondary pneumonia infections. It didn't help that research during the 1918 epidemic was mostly rushed and sloppy.
But then, four years later, two researchers at the Rockefeller Institute for Medical Research made an important discovery about influenza.
Experimenting on nasal washings from patients from the 1918 epidemic, they found that the disease could be transmitted to rabbits effectively, but no bacteria need be consistently present in the washings for the transmission to occur.2 In addition, the washings could be filtered through a filter small enough to remove all known bacteria, and the resulting filtered product could still cause disease.3 This showed that the infectious agent was something smaller than known bacteria, but still capable of replication and spreading to new hosts.
These researchers isolated a tiny rod-shaped organism, often small enough to fit through the filter, that they could grow in the lab; they named it Bacterium pneumosintes.4 Unfortunately, many other researchers could not replicate the results of Olitsky and Gates,5-7 though B. pneumosintes was later found to be a bacterial inhabitant of the human mouth and renamed Dialister pneumosintes,8,9 but the true agent of influenza was not discovered until the 1930s.
However, Olitsky and Gates went ahead and tried making a vaccine using this organism they discovered. They grew up two strains of it, one isolated from a patient in 1918 and one from a patient in a flu epidemic in 1922, killed these cultures with heat, and injected them into rabbits. The rabbits tolerated the vaccines pretty well, having some redness and swelling after the second of three doses.
When challenged with infectious bacteria, the results were as follows: two vaccinated rabbits and two controls were challenged with samples from rabbits previously infected with B. pneumosintes from previous experiments. The two controls got sick, while the two vaccinated did not.
Then another 17 vaccinated rabbits and 17 controls were challenged with cultures of B. pneumosintes, and 15 of the vaccinated rabbits did not get sick while the others did. So 88% effective. The two vaccinated rabbits that did get sick, and 10 of the ones that didn't, were also challenged with bacteria that cause secondary infections in flu patients (pneumococcus, Streptococcus haemolyticus, etc), and the 10 that didn't get sick were also protected against infection with these organisms, while the 2 that did were not, and succumbed.
So this shows, perhaps, that B. pneumosintes is indeed a pathogen, in rabbits at least, and a vaccine made from it protects rabbits from infection with it. Too bad it's not actually influenza.
Citations:
1. Olitsky, P. K. & Gates, F. L. Experimental Studies of the Nasopharyngeal Secretions from Influenza Patients X. the Immunizing Effects in Rabbits of Subcutaneous Injections of Killed Cultures of Bacterium pneumosintes. J Exp Med 36, 685–696 (1922).
2. Olitsky, P. K. & Gates, F. L. J Exp Med. 1921 January 31; 33(2): 125–145.
3. Olitsky, P. K. & Gates, F. L. J Exp Med. 1921 February 28; 33(3): 361-372.
4. Olitsky, P. K. & Gates, F. L. Experimental Studies of the Nasopharyngeal Secretions from Influenza Patients IV. Anaerobic Cultivation. J Exp Med 33, 713 (1921).
5. Andrewes, C. H., Laidlaw, P. P. & Smith, W. Influenza: Observations on the Recovery of Virus from Man and on the Antibody Content of Human Sera. Br J Exp Pathol 16, 566–582 (1935).
6. Garrod, L. P. Filter-Passing Anaerobes in the Upper Respiratory Tract. Br J Exp Pathol 9, 155–160.1 (1928).
7. Wilson, G. S. An Attempt to Isolate Bacterium pneumosintes from Patients Suffering from Influenza. The Lancet 209, 1123–1124 (1927).
8. Willems, A. & Collins, M. D. Phylogenetic Placement of Dialister pneumosintes (formerly Bacteroides pneumosintes) within the Sporomusa Subbranch of the Clostridium Subphylum of the Gram-Positive Bacteria. Int J Syst Bacteriol 45, 403–405 (1995).
9. Ghayoumi, N., Chen, C. & Slots, J. Dialister pneumosintes, a new putative periodontal pathogen. Journal of Periodontal Research 37, 75–78 (2002).
Previous research had suggested bacterial pathogens as the culprit, such as Pfeiffer's bacillus (aka Bacillus influenzae, later named Haemophilus influenzae) or others, but these could not be reliably found in flu patients, nor did serum from patients always contain antibodies against the bacteria. Vaccines made from them (such as in 019 or 026) didn't reliably protect against the flu, though they may have helped with secondary pneumonia infections. It didn't help that research during the 1918 epidemic was mostly rushed and sloppy.
But then, four years later, two researchers at the Rockefeller Institute for Medical Research made an important discovery about influenza.
Experimenting on nasal washings from patients from the 1918 epidemic, they found that the disease could be transmitted to rabbits effectively, but no bacteria need be consistently present in the washings for the transmission to occur.2 In addition, the washings could be filtered through a filter small enough to remove all known bacteria, and the resulting filtered product could still cause disease.3 This showed that the infectious agent was something smaller than known bacteria, but still capable of replication and spreading to new hosts.
These researchers isolated a tiny rod-shaped organism, often small enough to fit through the filter, that they could grow in the lab; they named it Bacterium pneumosintes.4 Unfortunately, many other researchers could not replicate the results of Olitsky and Gates,5-7 though B. pneumosintes was later found to be a bacterial inhabitant of the human mouth and renamed Dialister pneumosintes,8,9 but the true agent of influenza was not discovered until the 1930s.
However, Olitsky and Gates went ahead and tried making a vaccine using this organism they discovered. They grew up two strains of it, one isolated from a patient in 1918 and one from a patient in a flu epidemic in 1922, killed these cultures with heat, and injected them into rabbits. The rabbits tolerated the vaccines pretty well, having some redness and swelling after the second of three doses.
When challenged with infectious bacteria, the results were as follows: two vaccinated rabbits and two controls were challenged with samples from rabbits previously infected with B. pneumosintes from previous experiments. The two controls got sick, while the two vaccinated did not.
Then another 17 vaccinated rabbits and 17 controls were challenged with cultures of B. pneumosintes, and 15 of the vaccinated rabbits did not get sick while the others did. So 88% effective. The two vaccinated rabbits that did get sick, and 10 of the ones that didn't, were also challenged with bacteria that cause secondary infections in flu patients (pneumococcus, Streptococcus haemolyticus, etc), and the 10 that didn't get sick were also protected against infection with these organisms, while the 2 that did were not, and succumbed.
So this shows, perhaps, that B. pneumosintes is indeed a pathogen, in rabbits at least, and a vaccine made from it protects rabbits from infection with it. Too bad it's not actually influenza.
Citations:
1. Olitsky, P. K. & Gates, F. L. Experimental Studies of the Nasopharyngeal Secretions from Influenza Patients X. the Immunizing Effects in Rabbits of Subcutaneous Injections of Killed Cultures of Bacterium pneumosintes. J Exp Med 36, 685–696 (1922).
2. Olitsky, P. K. & Gates, F. L. J Exp Med. 1921 January 31; 33(2): 125–145.
3. Olitsky, P. K. & Gates, F. L. J Exp Med. 1921 February 28; 33(3): 361-372.
4. Olitsky, P. K. & Gates, F. L. Experimental Studies of the Nasopharyngeal Secretions from Influenza Patients IV. Anaerobic Cultivation. J Exp Med 33, 713 (1921).
5. Andrewes, C. H., Laidlaw, P. P. & Smith, W. Influenza: Observations on the Recovery of Virus from Man and on the Antibody Content of Human Sera. Br J Exp Pathol 16, 566–582 (1935).
6. Garrod, L. P. Filter-Passing Anaerobes in the Upper Respiratory Tract. Br J Exp Pathol 9, 155–160.1 (1928).
7. Wilson, G. S. An Attempt to Isolate Bacterium pneumosintes from Patients Suffering from Influenza. The Lancet 209, 1123–1124 (1927).
8. Willems, A. & Collins, M. D. Phylogenetic Placement of Dialister pneumosintes (formerly Bacteroides pneumosintes) within the Sporomusa Subbranch of the Clostridium Subphylum of the Gram-Positive Bacteria. Int J Syst Bacteriol 45, 403–405 (1995).
9. Ghayoumi, N., Chen, C. & Slots, J. Dialister pneumosintes, a new putative periodontal pathogen. Journal of Periodontal Research 37, 75–78 (2002).
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