Warning and Link to Worthwhile Read

I should mention, it may be a while until my next real post, because I'm working a big one. Maybe not big as in long, but as in the amount of work that's going into it. It's a real issue. So you can look forward to it, at least.

In the meantime, check out this well-written anecdote about a person that embodies (in a good way) pretty much all that is wrong with the anti-vaccine movement.

039 - Antipneumococcic Immunity Reactions in Individuals of Different Ages

A bit different today, not exactly about vaccines, but related: this study is concerned with immunity to pneumococcus and how that may change with a person’s age.¹

Sutliff and Finland, the study’s authors, tested the immunity of 134 subjects between the ages of 0 and 83 (some had just been born). They did a number of tests to determine immune responses to three types of pneumococcus (I, II, and III) or against pneumococcus as a whole species. These tests included analyzing the ability of blood to kill the bacteria or to protect mice against infection, and skin reaction tests when inoculated with type-specific polysaccharides or species-specific proteins or other cell contents.

Subjects were tested in hospitals, though for conditions unrelated to respiratory infection. Infants up to 2 were mostly not sick at all, just there for feeding. From 2-6 years, subjects were usually recovering from diphtheria or scarlet fever.

Sutliff and Finland found that, consistently through all type-specific tests, infants up to a couple weeks old had antibodies against all three types of pneumococcus (though most against type II and least against type I), but this immunity disappeared by 3-5 weeks after birth. Interestingly, whether a given infant had antibodies or not correlated very well with the immune status of their mother—if the mother had antibodies, chances are the infant did too, for a couple weeks.

Beyond 3-5 weeks, the proportion of subjects with antibodies gradually grew, so that by 2-11 years it was back up to the same level as among newborns. It did fall somewhat with advanced age though.

The species-specific tests didn’t seem to have any interesting pattern; they just increased as age increased.

So it seems that mothers can transfer their immunity to their offspring, at least to some extent for a short time after birth! Possibly through the placenta, if no other way. But this immunity fades, leaving infants susceptible—and indeed, the groups most susceptible to pneumonia were infants and the elderly.

It wasn’t really clear why immunity increased with age, since the subjects had never had pneumonia; possibly the subjects were exposed to some extent but not enough to become sick, or possibly they were asymptomatic carriers of pneumococcus. It was inconclusive.

Later studies citing this one had some interesting comments. Many interpreted Sutliff and Finland’s results as evidence that infants and young children don’t produce antibodies in response to pneumococcal exposure, which has obvious implications for vaccination efforts.^2–5 (Several other studies support this conclusion.)

Other comments:

 "Although earlier investigators had suggested that pneumococcal antibody declines with advancing years [039, this study], our studies showed an equal prevalence of IgG to PPS in our middle and elderly groups, probably reflecting the dynamic interaction of continued reexposure to S. pneumoniae and cumulative life experience, on the one hand, and antibody attrition together with a possible decline in antibody-forming capacity with aging, on the other."⁶

 "Invasive diseases caused by encapsulated bacteria including...pneumococci occur with highest frequency in infants and children after the decline of maternally derived antibodies."⁷

 "Sutliff & Finland state that immune bodies to the pneumococcus are present in the blood of infants immediately after birth and disappear about the end of the first month. These early type-specific antibodies are similar to those of the mothers, and are probably acquired by placental transmission."⁸

Citations:

1.  Sutliff, W. D. & Finland, M. Antipneumococcic Immunity Reactions in Individuals of Different Ages. J. Exp. Med. 55, 837–852 (1932).

2.  Weintraub, A. Immunology of bacterial polysaccharide antigens. Carbohydr. Res. 338, 2539–2547 (2003).

3.  Lindberg, A. A. Glycoprotein conjugate vaccines. Vaccine 17, Supplement 2, S28–S36 (1999).

4.  Lindberg, A. A. Polyosides (encapsulated bacteria). Comptes Rendus Académie Sci. - Ser. III - Sci. Vie 322, 925–932 (1999).

5.  Prevention of pneumococcal disease in sickle cell anemia. J. Pediatr. 129, 788–789 (1996).

6.  Musher, D. M. et al. Antibody to Capsular Polysaccharides of Streptococcus pneumoniae: Prevalence, Persistence, and Response to Revaccination. Clin. Infect. Dis. 17, 66–73 (1993).

7.  Schneerson, R. et al. Serum antibody responses of juvenile and infant rhesus monkeys injected with Haemophilus influenzae type b and pneumococcus type 6A capsular polysaccharide-protein conjugates. Infect. Immun. 45, 582–591 (1984).

8.  Guthrie, K. J. & Montgomery, G. L. Seven-Year Study of Pneumococcus Type Incidence in the Royal Hospital for Sick Children, Glasgow. J. Hyg. (Lond.) 46, 123–128 (1948).

038 - Immunization Against Vaccinia by Non-Infective Mixtures of Virus and Immune Serum

So about smallpox again. Obviously in full-blown smallpox, the skin lesions (see here) can cause some unsightly scarring. But even with the vaccine, an infection of cowpox/vaccinia virus can cause a few lesions and scars where it is inoculated into the skin, and this may not be ideal. So people wanted to see if they could improve the process and prevent this scarring.

One idea going around was, instead of using active virus by itself, mix it with serum from something already immunized with it, which would contain anti-vaccinia antibodies. This could prevent the virus from causing too bad an infection, while still allowing it to induce a good immune response.

So that's what today's study attempted.¹ C.P. Rhoads immunized 10 rabbits plus another 4 and with a mixture of vaccinia virus and serum from other rabbits that had been infected with vaccinia previously, so it was full of antibodies. Some other rabbits were kept un-vaccinated as negative controls.

Another interesting thing was that the test rabbits were inoculated by putting the mixture in their nose, rather than scraping it into the skin as usual. This was an unusual approach, so they had other rabbits inoculated in the skin the normal way as positive controls.

None of the rabbits immunized with the mixture had a negative reaction against the vaccine. Another rabbit got a vaccine of pure virus, no antibodies mixed in, and it produced the typical lesion that the virus makes.

So then the rabbits were challenged with the virus, and as expected, the negative controls produced the typical lesion that lasted a few weeks, but those immunized with mixtures previously only showed a small red spot that went away in 2 days.

Overall, the rabbits that received the mixture seemed to have good protection without a problematic response to the vaccine. Rhoads mentioned that it was important to have the correct proportions in the mixture though: too much serum and the vaccine wouldn't work well, but too little and the virus would form lesions as it normally did. Also, storing the mixture in a refrigerator was no good, since the virus escaped from the antibodies' clutches and wreaked its typical havoc.

Overall it seems interesting, but perhaps not very useful, especially if long-term storage isn't possible. And according to later studies, it might've been a misinterpretation somehow:

"The present work shows that a typical delayed hypersensitivity reaction in the absence of circulating antibody can be produced against vaccinia virus... Similar observations had been made in 1931 by Rhoads... The interpretation of his experiments was not clear at the time."²

Citations:
1. Rhoads, C. P. Immunization Against Vaccinia by Non-Infective Mixtures of Virus and Immune Serum. J Exp Med 53, 185–193 (1931).
2. Turk, J. L., Allison, A. C. & Oxman, M. N. Delayed Hypersensitivity in Relation to Vaccination and Multiplication of Vaccinia Virus in the Guineapig. The Lancet 279, 405–407 (1962).

037 - Relation of Vaccinal Immunity to the Persistence of the Virus in Rabbits

I once heard a claim from an anti-vaccine proponent that immunity from vaccines, instead of clearing the pathogen from the body the way the immune system does in the course of a “natural” infection, instead forces pathogens such as the measles virus to remain latent in cells, hiding in our bodies in an invisible state, until such time as our immunity might wane and they can be released to cause havoc. While I don’t think this claim was well-supported by any evidence, my encountering it did cause the current study to pique my interest.¹ It may not be very satisfying to my curiosity about the above claim though.

Some background: what was apparently a new technology, important at the time and foundational for this study, was electrophoresis (called cataphoresis by the authors Olitsky and Long): this technique, still used today mostly for separating all the proteins or nucleic acids in a sample based on their size, involves putting the sample into a gel immersed in a buffer full of electrolytes, and then running an electric charge from one side of the gel to the other. Since proteins and nucleic acids have an ionic charge, the current will pull them to one electrode or the other, depending on their charge. The larger ones get caught and stuck more as they’re pulled through the gel, so they move more slowly, while smaller ones can fit through smaller spaces, so they move more quickly.

In this study, though, their samples were whole viruses, rather than individual proteins or nucleic acids. People were trying electrophoresis on all kinds of different pathogens (viruses, but also protozoan parasites and virus-like bacteria^2–5) to see whether they moved toward the anode or the cathode. The technique herein was used to isolate and concentrate virus from animal tissues, virus that was too scarce to be detected by other methods (at the time). I should mention that in any case, “detection” of the virus involved inoculating other rabbits with the sample and seeing if they produced a characteristic infection reaction. It wasn’t good to be a rabbit in that lab in those days.

The hypothesis Olitsky and Long were testing was based on previous observations by others, that viruses could be detected in animal’s (and sometimes human’s) tissues longer after that organism had recovered from the viral illness. For example, polio virus had been detected from an infected monkey and a person several months after they had recovered. The authors speculated that immunity to a virus depends on the persistent presence of that virus in an animal’s tissues; after the virus is completely gone, the animal is no longer immune. (This observation and hypothesis didn’t distinguish between a “natural” infection or a live attenuated immunization, such as vaccinia inoculation as a vaccine against smallpox, so it wouldn’t support the anti-vaccine claim above in any case.)

Their model for this study was vaccinia (cowpox) virus infecting the testicles of rabbits. They took rabbits that had been infected at various times past, from 12 to 133 days previous to the study, and recovered. Samples of these animals’ tissues were taken under anesthesia, keeping the animals alive so they could be tested for immunity later, and electrophoresis was used to detect virus in the tissue samples. Olitsky and Long detected virus in samples from each of these rabbits, even the one infected more than 4 months before. (Note of contention: I don’t think they had any negative controls to make sure they weren’t just detecting contamination or something.)

Then the authors took five more rabbits that had recovered from viral infection 119 to 183 days before and tested them for virus. They took samples, seemingly randomly, from spleen, testicles, and skin, and checked for virus using electrophoresis. In the rabbits infected earlier, no virus was isolated, and they appeared to have lost their immunity; in those infected later, the authors did detect virus, and the rabbits seemed immune. So they concluded that the persistent presence of virus was necessary for immunity.

The next step was based on another previous finding that immunity seemed to be transmissible from mother to offspring. This was based on some observation of a pregnant mother and her newborn, and some preliminary animal study. So Olitsky and Long tested this hypothesis in two pregnant rabbits, one infected soon before giving birth, and the other infected but given time to recover before birth. The former’s offspring seemed immune, and electrophoresis recovered virus from the testicles of a male baby rabbit; but the latter’s offspring seemed susceptible to infection as normal. So the conclusion was that immunity (and persistent infection) is heritable only when the mother is infected at the time of birth.

These are interesting results, but the quality of evidence seems pretty low. There weren’t very good controls, and the sample sizes were quite small. Also it is in rabbits, which isn’t necessarily a good model animal. This poor quality seems borne out by later studies, some by the same researchers, that contradicted or found better explanations for these results:

"By the means employed in this investigation, vaccinal immunity has been shown to endure beyond the persistence of recoverable virus."⁶ (Similar results in other studies^7,8)

"Whatever the detailed mechanism, it is evident in our study that newborn rabbits have been made tolerant immunologically toward the antigens of vaccinia virus for at least a relatively brief period. Perhaps tolerance also explains the early observations of Olitsky and Long [037, this study] made on a single pregnant rabbit and its 3 offspring."⁹

References:

1.  Olitsky, P. K. & Long, P. H. Relation of Vaccinal Immunity to the Persistence of the Virus in Rabbits. J. Exp. Med. 50, 263–272 (1929).

2.  Olitsky, P. K., Rhoads, C. P. & Long, P. H. The Effect of Cataphoresis on Poliomyelitis Virus. J. Exp. Med. 50, 273–277 (1929).

3.  Kligler, I. J. Recovery of Fowl-pox Virus from Vaccines by Cataphoresis. Br. J. Exp. Pathol. 12, 42 (1931).

4.  Kligler, I. J. & Olitzki, L. Cataphoresis Experiments with Typhus Virus. Br. J. Exp. Pathol. 12, 69 (1931).

5.  Salle, A. J. The Electrical Behavior of Leishmania donovani. J. Infect. Dis. 49, 450–454 (1931).

6.  Morgan, I. M. & Olitsky, P. K. A Study of Immunity in Rabbits from Two to Three Years After Infection with Vaccine Virus with Attempts to Recover Active Virus. J. Immunol. 39, 1–15 (1940).

7.  Pearce, J. M. The Persistence of Vaccine Virus in Rabbits Immunized to Vaccinia by a Previous Infection and the Relationship of Immunity to Latent Virus. J. Infect. Dis. 66, 130–137 (1940).

8.  Olitsky, P. K. & Casals, J. Concepts of the Immunology of Certain Virus Infections. Bull. N. Y. Acad. Med. 21, 356 (1945).

9.  Flick, J. A. & Pincus, W. B. Inhibition of the Lesions of Primary Vaccinia and of Delayed Hypersensitivity Through Immunological Tolerance in Rabbits. J. Exp. Med. 117, 633–646 (1963).

036 - Effect of Calmette's BCG Vaccine on Experimental Animals

Back in 1929, there wasn't a very good vaccine against tuberculosis, though many people (including famous ones) had tried to make one. The one that seemed to have the most potential was called BCG, or Bacillus Calmette-Guérin, discovered by Calmette and others. (Modernity note: the situation hasn't really changed much since then. BCG is still the TB vaccine of choice, and doesn't work very well. Used sometimes in countries with endemic TB, not in the USA.) This is made from a strain isolated from a cow, and is the bovine version of tuberculosis, so isn't very virulent for other species (or for cows either, apparently). And after Calmette grew it in the lab for a while, it was even less virulent.

Calmette had tried it in thousands of infants since 1921, but hadn't kept good records, so the results are unavailable, unfortunately. But it seemed to produce mild TB consistently in lab rodents, thus protecting them from more serious disease.

This study tried to clarify some of the methods and results using the BCG vaccine in several kinds of animals.  Specifically: guinea pigs, calves, and monkeys, inoculated orally, subcutaneously, or intraperitoneally (near the organs).

Feeding BCG to animals generally didn't work very well: pigs and calves didn't show any tubercles indicative of tuberculosis response, though almost half the monkeys they tested did. And animals challenged with a virulent strain of TB after oral BCG vaccine got TB almost as much as controls, especially pigs and monkeys.

Subcutaneous vaccination worked a bit better. Many pigs showed tubercles and one may even have died from it; most monkeys and calves also showed an effect. Generally it was mild though. When challenged with a virulent strain, vaccinated guinea pigs generally had milder TB (or none at all) compared to controls; similar with monkeys, and I couldn't really tell with calves. So I guess that's a positive result, if it's the best you can get.

They got similar results with intraperitoneal vaccination in pigs, though the vaccine seemed rougher for them. But they seemed to have a little protection.

Overall, it didn't seem like great results, but it wasn't a great study.

Citation: King, M. J. & Park, W. H. Effect of Calmette’s BCG Vaccine on Experimental Animals. Am J Public Health Nations Health 19, 179–192 (1929).

035 - Antirabic Immunization with Desiccated Vaccine

People had been vaccinating against rabies for a while (at least since 1893), using nervous tissue from animals infected with an attenuated strain of the virus, but apparently this wasn't always perfect, and could be difficult to obtain.

Some people had been experimenting with preserving rabies virus by freeze-drying it. They found that this method could preserve the virus's infectivity for months or even years. But in the current study, D.L. Harris found that even after the freeze-dried virus lost its infectivity, it still retained some of its immunogenicity, the ability to induce an immune response. This disappeared after a while too, though, so there was a range in which it could be used as a vaccine. However, since there was a chance that it was still slightly infectious, it had to be given in conjunction with the regular vaccine to make sure there was no chance of contracting rabies from it; a sort of vaccine against the vaccine.

This doesn't seem all that useful to me, but apparently Harris went ahead and tried it on about 3500 people and a bunch of dogs and other animals. The people had all been bitten by a potentially rabid animal, so they didn't have much choice for treatment, and apparently the vaccine worked just fine in all except one, who had received a nasty bite but refused the full course of treatment so he could get back to work. The side effects in people were limited to short-lived local redness and swelling.

It seemed to work well prophylactically in animals too: of hundreds of dogs and some rats, only one rat died in the study but it wasn't clear that it was from rabies, and it seemed to be 100% effective in protecting rats against infection (of 16 vaccinated and 8 controls, zero got rabies of the former and 4 of the latter died of it).

So according to Harris, this vaccine gives better protection, but it seems like the disadvantage of requiring an extra dose of differently prepared vaccine makes it not quite perfect. More improvement needed.

Citation: Harris, D. L. Antirabic Immunization with Desiccated Vaccine. Am J Public Health Nations Health 19, 980–985 (1929).

034 - A Yellow Fever Vaccine

Back in 1928, yellow fever was causing problems in West Africa, causing epidemics and killing people who went there to investigate it. It’s caused by a virus that’s in the Flavivirus family, related to dengue and West Nile, and all these are transmitted by mosquitoes. The symptoms of yellow fever are usually fever, chills, nausea, muscle pain, and headache (among a few others), but sometimes it can go toxic and cause fatal liver damage. It’s classified as a hemorrhagic fever because it increases bleeding . Usually the mortality rate is 3% of cases, but can get as high as 50% in some outbreaks.

So, at a time in medical history when people were just learning that some infectious agents are smaller than bacteria, Edward Hindle isolated the yellow fever virus and attempted to make a vaccine against it.¹

He got the virus from a case of yellow fever in Dakar, Senegal. It was maintained by passing it through rhesus monkeys via mosquitoes; not a simple routine procedure, sounds like, not exactly streaking a Petri dish. But it worked: when he inoculated 16 other monkeys with only 1 μg of infected monkey liver, they uniformly died after a few days. It seems a lot more severe in these monkeys than in humans, but it makes for a good animal for vaccine tests I suppose.

So Hindle tried two different but similar ways to make a vaccine: the first used formaldehyde in saline to inactivate the virus, and the second used phenol in glycerin and water. In each case, some liver and spleen from an infected monkey was mixed with these ingredients, then filtered through a cloth and inoculated into test animals.

Hindle tested the formaldehyde version in one subject. It had a slight, short fever after immunization, but when inoculated with a dose of yellow fever that was about 2000 times more than a dose that would be lethal in most cases, the monkey only had a mild fever for 4 days. Two controls that received 1- or 10-times lethal doses both died as usual. Even a 10000-times lethal dose in the vaccinated monkey didn’t make it very sick.

So that’s pretty good, but the other was even better. The first monkey to get it had barely any reaction, if any, but resisted a 2000-times lethal dose with no symptoms, and then another 10000-20000-times dose.

Hindle was so pleased with these results that he tested another 10 monkeys (6 vaccinated, 4 controls). These 6 didn’t show any adverse reactions either. A second, blinded researcher inoculated the 10 with about a 10000-times lethal dose. All controls died as usual, but only one vaccinated died (who had actually received about 20000-times lethal dose); the other five were fine.

These seem like promising results, essentially a “yes or no” answer of whether the vaccine worked. There were controls involved and some blinding, though no placebo (not so important when doing an animal study though, perhaps). The author said human trials were worthwhile, though some tests should be done to determine the duration of immunity and the vaccine’s shelf-life.

However, future articles citing this study didn’t take such a bright view of it:

"Both preparations appeared to protect monkeys against yellow fever, but subsequent investigations of efficacy were inconclusive."²

"Overall, these preparations were problematic due to residual live virus or were ineffective due to inadequate antigenic potency, reflecting the rudimentary virological methods available at the time."³

"Results with this vaccine in human subjects were considered promising, though immunogenicity was described as irregular. Further attempts at refining and optimizing an inactivated YFV (yellow fever virus) vaccine however, were abandoned following the development of cost-effetive live attenuated yellow fever strains that were highly effective at protecting against natural YFV infection. These early attempts at developing an inactivated YFV vaccine were left behind as failures and this perception has persisted despite signs of early success."⁴

"The problems described with these vaccines were the presence of residual infectivity in viral preparations or the loss of immunogenicity."⁵

So I guess there are better YFV vaccines coming up in future posts.

Citations:

1. Hindle, E. A Yellow Fever Vaccine. Br. Med. J. 1, 976–977 (1928).

2. Hayes, E. B. Is it time for a new yellow fever vaccine? Vaccine 28, 8073–8076 (2010).

3. Monath, T. P. et al. Inactivated yellow fever 17D vaccine: Development and nonclinical safety, immunogenicity and protective activity. Vaccine 28, 3827–3840 (2010).

4. Amanna, I. J. & Slifka, M. K. Wanted, dead or alive: New viral vaccines. Antiviral Res. 84, 119–130 (2009).

5. Gaspar, L. P. et al. Pressure-inactivated yellow fever 17DD virus: Implications for vaccine development. J. Virol. Methods 150, 57–62 (2008).

033 - Immunization with Diphtheria Toxoid (Anatoxine Ramon)

So far we've seen the development of a promising diphtheria vaccine made of toxoid (inactivated toxin, 029 and 030), but haven't seen any trials to show that this vaccine actually protects its recipients from diphtheria. Unfortunately, today's study is not such a trial either.¹

The authors were comparing the Schick test for immunity to diphtheria toxin to a test that they had developed (in the future called the Moloney test). Both involved injecting a small amount of toxin under the skin of a patient to see if they reacted to it; if so, they were not immune. The main difference was the substance used as a control: in the Schick test, heat-inactivated toxin was used as a negative control, but Moloney and Fraser here used toxoid. This approach also helped determine whether an individual would have an adverse reaction to immunization with the toxoid at full strength.

So they did this test on 141 girls, ages 10-18 years. 47% were Schick-positive, reacting to the toxin but not toxoid control; 13% were Schick-negative, reacting to neither, and thus considered to be immune; 20% reacted to the toxoid control but not the toxin; and the last 20% reacted to both. Of this last group, it's hard to tell whether they're immune or not, but could be dangerous to immunize them.

So then the authors vaccinated the 47% Schick-positive subjects with toxoid, two doses of 0.5 mL each, a month apart. They did the Moloney test again after 1 1/2 months. Of those immunized, 65% became Schick-negative, 28% didn't, and 7% reacted to both toxin and control. They tested for antibodies in the latter group and found that half of them were immune. So the vaccine converted about 70% of Schick-positive to Schick-negative; thus 70% effectiveness, according to this test.

The authors concluded that their test was reliable, though the 70% effectiveness was not great for a vaccine. Perhaps three doses would work better than two.

Papers citing this study generally acknowledged the Moloney test as a useful thing. Here are some things they say:

"The use of diluted toxoid as a control interferes with the reading of the [Schick] test by producing more marked pseudo reactions, but is therefore of great value in indicating those persons susceptible to toxic symptoms following immunizing injections of antigen."²

"The writer is of opinion that, if one test has to be omitted, that test should certainly be the Schick-control and not the Moloney injection. It has been shown that the one test cannot be used as a substitute for the other, and of the two the information given by the Moloney test is much the more important."³

Citations:
1. Moloney, P. J. & Fraser, C. J. Immunization with Diphtheria Toxoid (Anatoxine Ramon). Am J Public Health (N Y) 17, 1027–1030 (1927).
2. Loeffel, E. & Massie, E. Relative Value of Heated Toxin and Toxoid as Controls in the Schick Test. Am J Public Health Nations Health 25, 1018–1022 (1935).
3. Underwood, E. A. The Diphtheria Toxoid-Reaction (Moloney) Test: Its Applications and Significance. The Journal of Hygiene 35, 449–475 (1935).

032 - Duration of Immunity Following Modern Smallpox Vaccine Inoculation

Safety and effectiveness are two important things with vaccines, but something else that is important to consider (that is also maybe part of effectiveness) is duration of immunity: how long someone is immune to a disease after being vaccinated against it. This varies considerably between different vaccines and diseases, depending on the strength and type of the immune response and on the pathogen itself, which may shift around its antigens so that the immune system no longer recognizes it (a good example of this is influenza).

So the study today is an attempt to get some handle on the duration of immunity to smallpox after inoculation with cowpox (aka vaccinia). Even in the days of Edward Jenner, some observed that people lost their immunity to smallpox sometime after being vaccinated. But the most specific they could say about that was that immunity lasted several years after one vaccination. It did seem that repeated exposure/inoculation led to more long-term immunity, such that if one were immunized three times about 5-6 years apart, immunity was life-long.

But in 1927, it was no longer legal to intentionally inoculate people with smallpox to test their immunity; at least not in Europe or the United States. No surprise. So researchers had to find other ways to determine immunity duration. Previous reports from Germany and Britain stated that immunity lasted 10 years on average, at which point it should be renewed, though a study in Algeria found a duration of only 5 years.

This article goes into some depth about proper vaccine technique and what reactions to expect when it's done right or not done right. Apparently many practitioners in the US were doing it wrong, in ways that had been banned in Europe. But when done right, the reaction observed in the patient's skin was an indication of how much immunity they had to cowpox before being inoculated, whether no immunity, partial, or full immunity. Though it wasn't clear exactly how much immunity to vaccinia paralleled immunity to smallpox; definitely a significant amount, of course.

So using these reactions, researchers tested the immunity of schoolchildren in California, inoculating them once in 1921 and then again in 1927, 6 years later. After the first round, the reactions indicated that about 55% had no immunity before inoculation (no surprise again), 12% had partial immunity, and 23% had full immunity somehow. The remainder didn't show any reaction from the vaccine, probably indicating poor vaccination technique.

Then after 6 years, they re-inoculated and observed reactions. If the vaccine worked well and immunity lasted at least 6 years, then perhaps 10% of subjects should show no or partial immunity from the re-test, while the rest should be fully immune. But actually, 1% had no immunity, 26% had partial, and 73% had full. So 15-25% of subjects had declined in their immunity after 6 years from the first inoculation.

This wasn't a great study by any means, but it was something, and should've been helpful in establishing a schedule for re-inoculation. Progress!

Citation: Gillihan, A. F. Duration of Immunity Following Modern Smallpox Vaccine Inoculation. Am J Public Health (N Y) 17, 906–911 (1927).

031 - The Value of Mixed Vaccines in the Prevention of the Common Cold

You can probably guess from the title what the conclusion of this study was.

That said, it was actually a very good study, at least for the time; future studies claimed that this one might've been the first real clinical trial. It was in the vein of post 026, using a vaccine made of multiple types of killed bacteria to immunize against the common cold.

This one took place at the University of Manchester, and contained four main groups of subjects: those that volunteered to be randomly assigned either to vaccination group or control group, those that volunteered to be vaccinated, and those that volunteered to be controls. So about half the subjects were randomized and the others weren't. This didn't make much difference in the end though. The randomized groups, at least, were about evenly matched in sex and time since last cold.

The vaccine was made with several different types of bacteria commonly found in respiratory infections, including pneumococcus. Three injections were given, a week apart. Most reactions, if any, were trivial; some had an induration lasting a couple weeks, and one had a swelling that became an ulcer that healed slowly. But nothing very serious.

However, considering the results, the risks probably weren't worthwhile: the vaccinated groups got sick more than the controls. It wasn't a significant difference, 1.47 colds per person for vaccinated compared to 1.14 per person for controls, but it was quite clear that the vaccine did not help. Severity of illness was no less for the vaccinated group either. And the pattern was the same for the randomly assigned groups as for those that volunteered for one group or the other.

The scientists kept good records of different things in this study, and considered the possibility of different kinds of bias, so it was a relatively good study overall. There was randomization and group matching and such. Not up to today's standards, of course—it wasn't single- or double-blinded and there wasn't a placebo, for two things—but it was progress.

As a side note, there was a graph of how many colds lasted how long, and most of them were around 7 days long (as expected), though a few were more than 50 days.

Finally, I liked this quote from the article:

"We would add a word of warning with regard to the utter uselessness of the reports of individual patients as evidence of efficient prophylaxis. Among any large population, some persons will experience fewer colds during any particular winter, than they have experienced in previous year. In no department of human reasoning does the argument, post hoc propter hoc hold more absolute sway, than in the lay evaluation of medical procedures."

Always a good thing to remember, regarding conventional medicine or alternative medicine alike.

Citation: Ferguson, F. R., Davey, A. F. C. & Topley, W. W. C. The Value of Mixed Vaccines in the Prevention of the Common Cold. J Hyg (Lond) 26, 98–109 (1927).

030 - Diphtheria Immunization with Diphtheria Toxoid (Anatoxin-Ramon)

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).

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).

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:

"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).

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).

026 - VII. Report on the Prophylactic Vaccination of 1536 Persons Against Acute Respiratory Diseases, 1919–1920

Considering the difficulties of previous prophylactic vaccine studies, especially during the 1918 flu pandemic, there was something of a movement to improve the quality of future studies. Two things were the focus in particular:

• previous studies had immunized during the pandemic, so there was a question of whether immunity had been established in time before the subject was exposed; it would be better to immunize long before the expected exposure, and
• previous studies' control groups hadn't always been very similar to the experimental groups in size or demographics, so it was difficult to compare the populations.

So some tried to do better. In the current study, vaccines were made to try to protect against all known causes of respiratory disease (which known causes, at the time, were all bacterial), such as pneumococcus. The subjects were employees at the Metropolitan Life Insurance Company (MetLife) in New York City. Those who volunteered, 1536 in total, received a vaccine; 1293 got three doses, the rest only one or two (probably due to side effects). There were another 3025 employees as controls, who got nothing. In order to ensure that the two groups were similar, the scientists administered questionnaires about personal history, home conditions, and habits, and the groups did seem pretty similar in sex, age, and habits.

In terms of side effects, there were the common local (redness, soreness) and general (headache, malaise) ones; a few people got fever and such, but nothing was very alarming.

For results, one impressive thing was that vaccinated employees were absent about half a workday less than controls, on average.

Other than that, the results were not very impressive: for almost all diseases, the inoculated group had more cases than the control group, sometimes more than twice as many. The main exception to this was pneumonia: there was one case in the vaccinated group (0.05%) in a girl who had only one of the three recommended doses, compared to 11 cases (0.29%) in the control group. That's a five-fold difference.

The authors said, "One might infer from the tabulated figures that it were wiser not to be inoculated." They make a good point, though, by reminding the reader that the vaccinated subjects had volunteered for it, and were thus more likely to be more susceptible to respiratory infections. Indeed, according to questionnaires, about 22% of the controls said had been fairly free of infection, while only 10.5% of the volunteers said the same.

So overall, it seemed that the vaccine helped against pneumonia and not much else, not surprisingly (since many respiratory infections are viral, not bacterial). This seems like a pretty good example of a negative study. With its accounting for previous history and attempting to ensure similarity between groups, this study seems better than the ones I've written about previously. Clinical trials seem to have taken a step forward at this point. But there was still no blinding, placebo, or randomization, so there's a lot of improvement yet to be made. Right now it's still pretty much correlational (an observation of people who choose to be vaccinated compared to those who don't). So we'll see.

Citation: Von Sholly, A. I. & Park, W. H. VII. Report on the Prophylactic Vaccination of 1536 Persons Against Acute Respiratory Diseases, 1919–1920. J Immunol 6, 103–115 (1921).

025 - Mills-Reincke Phenomenon and Typhoid Control by Vaccine

This article wasn't really about the safety or effectiveness of a particular vaccine, but it does relate to the question of whether it's worthwhile to vaccinate the whole population (and also another concern people sometimes raise), so I thought it was interesting.

First off, a definition: The Mills-Reincke phenomenon (and similarly, Hazen's theorem) was, hypothetically at least, the idea that typhoid fever could be prevented by cleaning up a population's water supply (since typhoid is spread through fecal contamination of water and such), and that for every typhoid death prevented by cleaning the water supply, several deaths from other causes would also be prevented by the same sanitation efforts. Presumably these other deaths might be caused by other water-borne infectious agents, or a general weakening due to any kind of water-borne infection.

At the time, public health officials in the US were considering vaccinating the population, or at least certain parts of it, against typhoid. The author of this paper warns against that, saying that this approach may mask the true problem (contamination of the water supply). Instead, water should be made as clean as possible, and typhoid cases may be used as an indication of contamination.

To support this position, he presents evidence from Alpena, Michigan, a town situated near Lake Huron. This town took its water from the lake and also dumped its sewage therein, and not surprisingly suffered from typhoid fever. But in the years between 1900 and 1918, a few changes occurred in the water supply: first, in 1907, the water intake was moved to a more polluted section of the lake; then in 1915, improvements were made in the sterilization of the water.

As a result of these changes, the annual mortality rates (that is, number of people dying each year) from typhoid or all causes changed. The average mortality from typhoid between 1908 and 1915 was higher by about 36 deaths per 100,000 people than the average between 1900 and 1907; in contrast, the mortality from all causes was higher by almost 150 deaths per 100,000. So for every one extra typhoid death, there were about 3 more deaths from other causes, in line with Hazen's theorem.

And when the water was cleaned better in 1915, there were about 4 fewer non-typhoid deaths for each one typhoid death prevented, though the data for this period is limited to only two years (1916 to 1917; the 1918 flu epidemic kinda skewed the all-cause mortality data for that year).

So it does seem that, given a choice between vaccinating against typhoid vs. cleaning up the water supply, the latter is the better option. Not surprising. Lest anyone conclude that this makes vaccines less valuable, though, the average mortality from typhoid between 1916 and 1918 was still about 13 deaths per 100,000 people, so there was still room for vaccinating to be useful. At least back then. Since we don't really do typhoid vaccination much in the US today, I suppose it turned out not to be helpful. This study doesn't say much about any other kind of vaccine though.

Citation: McGee, H. G. Mills-Reincke Phenomenon and Typhoid Control by Vaccine. Am J Public Health (N Y) 10, 585–587 (1920).