Fred Neufeld and pneumococcal serotypes: foundations for the discovery of the transforming principle

Abstract

During the first decade of the twentieth century, the German bacteriologist Fred Neufeld, later Director of the Robert Koch-Institute in Berlin, first described the differentiation of pneumococci into serotypes on the basis of type-specific antisera. This finding was essential for subsequent research at the Rockefeller Institute of Medical Research (RIMR) in New York, and elsewhere, aiming for the conquest of human pneumococcal pneumonia, including antiserum therapy, the discovery that the type-specific antigens were carbohydrates, and the development of effective multivalent pneumococcal polysaccharide vaccines. Moreover, on the basis of pneumococcal serotypes Fred Griffith, in 1928 in London, discovered pneumococcal transformation, and Oswald T. Avery and coworkers, in 1944 at RIMR, identified DNA as the transforming substance. This sequence of events, leading to today’s knowledge that genes consist of DNA, was initiated by a farsighted move of Simon Flexner, first Director of the RIMR, who asked Neufeld to send his pneumococcal typing strains, thus setting the stage for pneumococcal research at RIMR. Here, we describe Fred Neufeld’s contributions in this development, which have remained largely unknown.

Introduction

Ever since Emil von Behring and Shibasaburo Kitasato had reported in 1890 that infected animals can be cured by antiserum of animals immunized with the corresponding bacterial toxins [1, 2], attempts were made worldwide to combat infectious diseases with immune sera. Two physicians, the brothers Felix and Georg Klemperer, observed as early as 1891 that animals inoculated with the sputum of pneumonia patients became immune, and that patient sera could protect animals against subsequent infection with “Fraenkel’s diplococcus” [3]. Later dubbed streptococcus pneumoniae, or pneumococcus, these bacteria had first been described 1886 by Albert Fraenkel as a cause of human pneumonia [4]. The Klemperers’ observations stimulated a great number of physicians and microbiologists, to engage in studies to develop a serum therapy for human pneumonia, then a leading cause of death. One of them was Fred Neufeld, at the time employed at the Robert Koch Institute but commissioned to the Kaiserliche Gesundheitsamt (Imperial Health Service) in Berlin. His main interest was in the emerging field of serology. As will be discussed in detail below, Neufeld, together with a colleague, Staabsarzt Dr. Haendel, were the first to discover pneumococcal serotypes, and to observe that protection by immune serum was type-specific. They published this in several papers during 1909 [5–7].

Since its foundation in 1910, physician-scientists at the Rockefeller Institute’s Hospital in New York have been engaged in studying the pressing problem of human pneumococcal pneumonia [8]. Simon Flexner, the first Director of RIMR, took note of Neufeld’s work on pneumococcal serotypes and, immediately realizing its importance, asked Neufeld to provide samples. Flexner urged Rufus Cole, Director of the Rockefeller Hospital, to hire Oswald T. Avery, then working at the Hoagland Laboratory under the direction of Benjamin White, a chemist by training. This experience prepared Avery in subsequent years to determine, with his colleague Michael Heidelberger, the pneumococcal antigens that induced protective immunity as carbohydrates [9, 10] and, together with Colin MacLeod and Maclyn McCarty, the chemical nature of the transforming principle as DNA [11].

Avery arrived at RIMR in 1913 and joined forces with Alphonse R. Dochez who had already began to study human immunity to pneumococci [12]. In 1915, Avery and Dochez reported initial studies of pneumococcal types and their relationship to human pneumonia [13], followed in 1917 by the Rockefeller Institute Monograph No. 7 in which Avery, Chickering, Cole, and Dochez presented a comprehensive and authoritative account of success in treating pneumonia with type-specific horse antiserum, an astonishing feat at the time [14].

It is no surprise that this report led to many efforts particularly in the USA to produce horse antisera and their use to treat pneumococcal pneumonia. The textbooks of medicine of that era as well as the first and second editions of Principles of Bacteriology and Immunity by Topley and Wilson noted reports of many clinical trials with mixed results [15]. Far too frequently, clinical trials reported little or no success with antisera therapy. Type I and Type II antisera were often successful but not Type IV and other types. Later, it was learned that the antibodies in certain horse antisera lacked phagocytic properties which was responsible for failure of antiserum therapy. Yet it is no surprise that there were hundreds of reports on efforts to produce effective antisera for therapy. The stakes were high. Pneumonia was one of the most common causes of death in that era.

After Michael Heidelberger and Avery reported that the type-specific pneumococcal antigens were polysaccharides, there were many efforts to develop a pneumococcal vaccine, all ending in failure. Success was not achieved until a clinical trial at a US Army military base in 1944 [16]. But for many technical practical reasons, the current highly successful pneumococcal vaccine was not developed until the 1970s.

The experiments that led to the discovery of the transforming principle had originally been done with the aim to further unravel pneumococcal pathogenicity. McCarty in the chapter “The Sugarcoated Microbe”,1 in his book The Transforming Principle [17] remarks: “It is often pointed out that research in the basic sciences provides the base of new knowledge essential for the development of the applied sciences, including medicine. We are less frequently reminded that the reverse can also occur. From the initial discovery of the phenomenon known as ‘the transformation of pneumococcal types’ until the identification of the transforming substance as DNA, all of the researchers were medical bacteriologists primarily interested in the cause and control of human pneumonia” [17]. The history and the individuals involved have been extensively and thoroughly covered in the literature, notably in two books, one by Maclyn McCarty [17], the other by René Dubos [18], as well as elsewhere [19, 20]. We will make no attempt to repeat this saga here, and only mention the highlights.2

Briefly, Avery and collaborators were intrigued by an experiment published in 1928 by Fred Griffith in the UK, in which Griffith showed that avirulent pneumococci could be transformed into virulent ones by coinoculation of mice with strains of both kinds [21]. Avirulent pneumococci, also referred to as R-forms because they grow as rough colonies on agar, arise during culture passages due to loss of the carbohydrate capsule. They are non-pathogenic because they are sensitive to enzymatic attack in the host. Virulent pneumococci, owing to their capsule, are protected from enzymatic attack and cause septicemia in the mouse. They are referred to as S-forms because they form smooth colonies.

Griffith reported that upon co-inoculation of a mouse with a live R-strain and an excess of heat-killed S-bacteria, live virulent S-bacteria can be recovered from the mouse that produce a capsule and maintain virulence for many generations. Thus, avirulent non-encapsulated cocci had been transformed into virulent encapsulated ones. Moreover, if the coinoculated pneumococcal strains differed in serotype, the recovered live bacteria expressed the serotype of the heat-killed S-strain, not their own original serotype. Griffith concluded that the live avirulent cocci had engulfed material from the heat-killed virulent bacteria, a process that enabled them to express a novel carbohydrate capsule. He envisaged this material as “pabulum”, something to eat [21].

The objective of Avery and co-workers was to identify the chemical nature of the pabulum. To this end, they first developed an in vitro system that allowed them to transform avirulent cocci in a test tube, using extracts from virulent bacteria. They then fractionated the extract, using various methods available at the time. Depletion of proteins, lipids, or carbohydrates did not affect transforming activity. In contrast, a highly purified fraction that contained nucleic acids retained transforming potential. DNAse destroyed it, RNAse did not. They thus showed that Type II R-forms could be transformed into Type III S-forms using DNA from Type III pneumococci [11]. The results, exciting and far reaching as they turned out to be, were not directly relevant to the conquest of pneumonia, the objective of the Avery laboratory. McCarty remarked: “Admittedly, in the later stages of the search we came to see that our findings would not help to eradicate pneumonia” [17].

Pneumococcal serum therapy, the transformation of pneumococcal types, the development of pneumococcal vaccines, and the discovery of the transforming principle—all four rested heavily on Neufeld’s original discovery of pneumococcal serotypes. Although Neufeld’s work is to some extent quoted in the relevant literature, his name and contributions are mentioned only briefly and in passing [8, 17–19]. As a result, we felt that the groundbreaking role of Neufeld’s contributions as foundations of the subsequent developments has not been adequately appreciated.

When we took a closer look at Fred Neufeld, we detected not only a creative and farsighted scientist but also a man of upright character who combined professional and private integrity in difficult times.

Education and career

Fred Julius Neufeld was born on February 17, 1869 in Neuteich, a small village near Danzig, West Prussia, (now Novy Staw, Poland). He was the only son of Dr. Hermann Neufeld, a general practitioner, and his wife Johanna. The family was part of a Mennonite community that had settled in West Prussia in the sixteenth century. Because the family was moderately well to do, Neufeld could study medicine and passed his final examination in Heidelberg in 1892. A year later, he obtained his doctoral degree with the grade “insigni cum laude” (second best) with a written thesis on “Case Studies on Innate Tumors of the Scull” [22].

After a year of work as a physician, in 1894 Neufeld joined the Royal Prussian Institute of Infectious Diseases in Berlin as a deputy assistant without pay. This institute, in 1912 renamed Royal Institute of Infectious Diseases Robert Koch, is commonly referred to as the Robert Koch Institute (RKI). In 1897, Neufeld was promoted to staff assistent, meaning that he received a salary. His initial assignment was to work on cholera, but in his memoir he stated that “this plague was already close to having vanished at the time, (so that) I was searching for new fields of activity” [23].3 As a result, Neufeld turned to the study of typhoid, and in his first publication he proudly reported the detection of typhoid bacilli in tissue recovered from roseola lesions [24], raising some interest in the medical community [22, 23].

As of 1900, Neufeld became enrolled in Koch’s studies on malaria and tuberculosis, and in 1903 he accompanied Koch to Rhodesia to investigate East Coast Fever of cattle. He did not seem to have overly enjoyed the tropical experience: “Beginning of 1904 the work on the new disease had progressed sufficiently so that I could quit and return to Berlin to continue working on tuberculosis. On the side, I could concentrate on my serological interests” [23]. By now having been promoted to Professor, in 1904 Neufeld was commissioned as advisor to the Imperial Health Service in Berlin, and became fully employed there as a research officer from 1907 to 1912. Neufeld returned to the RKI in 1912, now as department head. When the post of director became vacant, Neufeld was offered the directorship, but only after three other candidates had declined. On Sept 1, 1917, Neufeld became Director of the RKI [22, 25–27].

When Neufeld became director, the Institute had six departments, with one of them, Bacteriology, led by Neufeld himself. Under Neufeld’s leadership the RKI expanded to 10 departments under varying names such as: Plagues, Tropical Diseases, Rabies, Chemistry, Medical Chemistry, Serology, Smallpox, Chemotherapy, etc. After WWI and during the Weimar Republic, it became more and more difficult to secure sufficient resources for research. Important work was still done, but less than before WWI. An Emergency Association (Notgemeinschaft) of German Science was established that provided research grants on a competitive basis. By far the largest individual grant was given to Neufeld for studies on BCG vaccination. Several RKI scientists including Neufeld were supported by grants from the Rockefeller Foundation, which kept an office in Paris for European applications. Efforts were made to maintain international relations: Prominent scientists from all over the world visited the RKI during these years, including Simon Flexner, Rufus Cole and Theobald Smith, all from RIMR. The role of the RKI as an educational center for foreign visiting fellows resumed after WWI by attracting particularly Japanese and Russian guest scientists. Although German scientists were still discriminated against at international conferences and commitees, Neufeld participated in the international serum standardization program of the League of Nations and attended the serum conferences in Paris 1922 and Geneva 1924 [22, 25–28].

Of far reaching consequence was the relationship of Neufeld and Simon Flexner, first director of the RIMR in New York. The relationship may have started in 1910 when Flexner asked Neufeld to provide samples of the pneumococcal serotypes that Neufeld had identified. A highlight of their friendship was Flexner’s invitation to Neufeld, in 1925, to visit him at RIMR in New York. Flexner arranged for a transfer of funds to Neufeld to pay for passage and travel expenses, and the visit took place in 1926. Neufeld gave a Harvey Lecture—an outstanding honour—and several other prestigious lectures at major US universities. Flexner returned Neufeld’s visit in 1927, an event that was covered by the New York Times and major German newspapers [29, 30] (Fig. 1).

Fig. 1.

Newspaper clipping found among the uncataloged Neufeld papers at the RKI [29]; photo taken during the visit of Flexner at the RKI in 1927

Neufeld’s Harvey Lecture, delivered in New York on October 30, 1926, was entitled “Origin and Dissemination of Tuberculosis According to Recent Investigations” [31]. Interestingly, in this and other lectures, Neufeld often critically reviewed the work of others, with minimal reference to his own contributions. Here, he referred to his former mentor Robert Koch who, in the ongoing controversy about the relationships of bovine and human tuberculosis, had proposed “…the view, held before him by Theobald Smith, that the infective agents of bovine and human tuberculosis are different from each other”. Neufeld felt obliged to mention “…the quite justifyable opposition which this statement in his London Lecture (of 1901) had aroused…”, not without stressing that Koch would have been more convincing had “…a clear differentiation between bovine and human tuberculosis…. been established and the means indicated whereby both types of bacilli could be distinguished…” [31]. We now know that, even without this evidence, Koch was right.

Nazi rule, resignation, and retirement

In early 1933, at age 64, Neufeld fell ill with what was referred to in his physician’s statement as a “severe influenza” (schwere Grippe) leading to a “considerable general state of exhaustion which will take several months to resolve” [29]. On March 1, Neufeld asked the Minister of the Interior for 3 months sick leave, and, on May 23, he wrote a letter of resignation. The reasons for this move have never been unequivocally determined, and have been the subject of much speculation. Here, we briefly summarise the circumstances preceding Neufeld’s resignation, for more detail the reader is referred to references [22, 25, 26], and further references cited therein.

The Nazis seized power in Germany on January 30, 1933. One of the first laws the Nazis passed was the “Gesetz zur Wiederherstellung des Berufsbeamtentums” (Law to reestablish the professional public service), which in fact was a law to discharge Jews from state employment. As a result, seven employed Jewish research scientists of the RKI were handed their dismissal documents on March 31, 1933, to become effective by June 30. A few days later, the date was changed to May 15, indicating how urgent the matter had to be handled. In addition to the seven staff scientists, at least three Jewish technical assistants and at least five voluntary (unpayed) Jewish junior scientists had to leave.

Neufeld as director of the RKI had hired most of the senior scientists during the pre-Nazi period, in spite of the anti-Semitism already prevalent at that time. They represented the promising talent of the RKI, such as Fritz Kauffmann, whom Neufeld had recommended to Flexner for a visit to the RIMR, and who was to become known for the Kauffmann–White scheme for serotyping salmonella strains. Moreover, internationally respected scientists including Walter Levinthal,4 who had received the Paul Ehrlich Prize, and who had worked at RIMR, Alfred Cohn, later professor at Yale, and Neufelds’s favorite coworker, Rochla Etinger–Tulczynska, were among those who had to leave. (For a full account of the names and their further fate, see [25].)

Neufeld learned of the discharge of his scientific staff during his sick leave. Friedrich Kleine, Neufeld’s deputy director and eventual successor and a member of the Nazi party, had signed the letters. It is most likely that Neufeld had no desire to keep directorial responsibility for a RKI that had discharged most of its leading scientific ranks. Significantly, he applied to retire as of July 1, the very date of dismissal of his colleagues [25, 26, 29].

In the US and England, it was suspected that Neufeld’s resignation was not entirely voluntary but forced by the Nazis. In several letters to people that offered to accommodate him if he were to leave Germany, Neufeld insisted that his illness was the only reason and that he had no difficulties with the authorities: “My retirement was voluntary and (as I have a clear Aryan pedigree) the only reason for it—and a sufficient reason it seems to me—was my disease. Otherwise, the government would not have made me a honorary member of the Institute” [25, 29].

Nevertheless, there are reasons to assume that the Nazi government was not altogether unhappy with Neufeld’s early retirement [22, 25, 26]. Neufeld had never joined the Nazi party, although everyone carrying official responsibilities in Germany was strongly encouraged to do so. Moreover, Neufeld was opposed to the prevailing attitude, favoured by the Nazis, that all microorganisms should be indiscriminately eradicated by all available means, and strongly criticized the exaggerated procedures of quarantine and disinfection then in use [32]. This was very much in contrast to his successor, Kleine, who was Hermann Göring’s favorite because of a “more appropriate, military, colonial, and microbe-hunting career” [33].5 In any case, in his letter of acceptance the Prussion Minister of the Interior, Göring, thanked Neufeld profusely for his engagement and informed him that he would be allowed to continue working at the RKI as a honorary member, and to use his laboratories and the “required facilities at no cost” [29]. The Rockefeller Foundation resumed supporting his work and Neufeld could continue his studies, but now without responsibilities for the RKI’s new policies.

The correspondence between Neufeld and Flexner continued until 1935, and there are no letters between them after that [29, 30]. Noted here are excerpts of an exchange of letters in 1935. Neufeld and Flexner had now retired as Directors.

Letter from Neufeld to Flexner, August 3, 1935:

“The Koch Institut is now being merged with the bacteriological division of the Reichsgesundheitsamt – which I do not consider fortunate….

I had the luck of having, – in Levinthal, Frau Etinger-Tulczynska and recently in Sevag6 who was born in Armenia but is an American citizen, – distinguished, original and gifted collaborators”.

Letter from Flexner to Neufeld September 5, 1935:

“It is sad to think of the Koch Institute losing its identity. It can never lose its place as the foremost institution of bacteriology in history, and some day it may come back again to renew its youth and purpose. I sincerely hope it may.….Levinthal seems to be doing well in London. I am very glad”.

What is clear in a review of these letters from 1910 to 1935 [29, 30] is that both men had great respect and affection for each other.

Neufeld was active in the laboraory until at least 1943 when he still published two papers. In 1944, at his 75th birthday, Neufeld was awarded the Goethe Medal, then the highest honour in arts and sciences in Germany. Neufeld died on April 18, 1945, during the final battle of WWII in Berlin. The obituary, written by Kleine, gave as the cause of death extreme fatigue and exhaustion [34].

Pneumocccal studies prior to serotypes

Neufeld began to work with pneumococci during his first period at the RKI when he had not yet decided to concentrate on one of several microbial infections under study there, including cholera, typhoid, tuberculosis, among others. But already in his first papers on pneumococci, he described two observations that were to become, albeit without Neufeld suggesting so, the basis of widely used diagnostic tests: the bile solubility of pneumococci and the Quellung reaction. His paper on bile solubility appeared in 1900, and described the specific lytic effect for pneumococci by the bile of several animals [35]. This lytic effect of bile became a standard bacteriologic test to distinguish pneumococci from streptococci. The latter were resistant to the lytic effect of bile.

Of still greater diagnostic importance was the Quellung phenomenon, first published by Neufeld in 1902, in a paper dealing with bacterial agglutination. Neufeld writes: “In all of these investigations Fraenkel’s pneumococci have received little attention. Indeed, if one understands agglutination as usual, i.e. as a process in which upon addition of a specific serum single microorganisms get clumped together in a big lump, then an agglutination in this sense….has so far never been described”. What rather happens is this: “Upon mixing of equal parts of agglutinating serum and pneumococcal broth culture,…..one observes in a hanging drop a distinct swelling (Quellung) phenomenon that, with a strong acting serum, occurs immediately or within a few minutes” [36]. The phenomenon became internationally known under the German term “Quellung Reaction”. To identify serotypes of pneumococci isolated from patients who received homologous serum therapy, Avery et al. [14] used a more rapid variant of the Quellung Reaction by testing peritoneal fluid of mice inoculated 4 h earlier with the patients’ sputum. To further speed up the selection of the proper serum for treatment, British investigators reported in 1932 that patient sputum could be used directly for pneumococcal typing by the Quellung reaction [37, 38]. Only then did Neufeld consider this reaction for rapid bacteriologic diagnosis [39].

Neufeld was more interested in developing a theory of agglutination on the basis of the Quellung phenomenon [36], a rather elegant one considering how little was known about antibodies. In the following papers, he and Walter Rimpau demonstrated that the immune defence against pneumococci (and streptococci) involved the combined action of antibodies (they suggested the term “bacteriotropins”) and phagocytes, but did not require complement [40, 41]. On that basis, they developed “the bacteriotropin theory” [41], which in fact represented the first unifying concept of the coordinated function of antibodies and phagocytes in anti-bacterial immunity. Neufeld and Rimpau thus felt that they had resolved the Ehrlich/Metchnikov conflict, for decades the dominating controversy in immunology on the roles of antibodies and phagocytes in immunity, i.e. between “humoral” and “cellular” immunity ([42], with further references therein). Although essentially correct, Neufeld’s and Rimpau’s ideas could not compete with those of the science giants Ehrlich and Metchnikov and their respective followers, and so the struggle went on for years to come. Neufeld states in his memoir: “It is amazing for how long the controversy went on between the ‘humoral’ conception of immunity, maintained in Germany under the impression of the discoveries of Behring, Pfeiffer, and Ehrlich, and the ‘phagocyte theory’, fostered in France by Metchnikov and his numerous disciples at Pasteur” [23].

Pneumococcal serotypes

In many of his papers [5, 36, 40, 41, 43], Neufeld described in much detail his methods to produce and test pneumococcal antisera. He was not the only one to try to generate potent sera for therapeutic purposes, and opinions differed widely about techniques and protocols. Two questions were of particular interest, the method of testing the antisera, and that of their “valency”. As to the test methods, Neufeld kept stressing the quantitative aspect, i.e. the need to carefully calibrate a serum by titrating it against a series of dilutions of the bacteria, in a mouse protection assay. Quite frequently, he criticized his competitors, if they had published results at variance with his own, for using inadequate analytical techniques.

Opinions on valency also varied widely. While most experts held the view that an antiserum against a single isolate protected against all pneumococcal strains, it was also proposed that an antiserum would protect only against the strain used for immunization [44, 45]. After discussing the situation, Neufeld and Haendel described their own observations: “Our experiments yielded the following: Using a univalent highly active serum against a single strain, as well as several patients’ convalescent sera, we observed protection not only against the homologous strain, but also against a series of other strains, and the protection was quantitatively not significantly lower. ….After we had obtained similar results with a series of cultures, we were surprised to see no effect of our serum on cultures isolated from two cases of pneumonia that clinically presented nothing unusual….Also there was no effect of our human convalescent sera”. They considered the existence of “serum-fast” pneumococci, but rejected the possibility because they found that the post-crisis serum of one of the patients protected mice against pneumococci of both atypical isolates [5].

The latter finding was the starting point for a comprehensive clinical study on the role of antibodies in the recovery of human patients from pneumonia, as manifested in the dramatic drop of temperature, known as the “crisis”. The first aim of the study was to prove that sera taken after crisis contained protective antibodies, which was a controversial issue at the time. Neufeld and Haendel found antibodies in every case: “We not only became convinced that the pneumonic crisis is exclusively due to antibodies, but that these antibodies are also experimentally demonstrable….Above all, the antibodies found in the blood of convalescents behave exactly like those artificially generated in experimental animals….” [6].

In a continuation of this study, Neufeld and Haendel identified three pneumococcal serotypes [6, 7]. Here, we reproduce only one of a series of experiments in which they showed that an antiserum against the prototype strain, termed “Pneum. I”, did not protect against strain “Pneum. Franz”, named after the patient from which it was isolated, and vice versa. Moreover, pneumococci from two other patients (“Pneum. Br.”, “Pneum. Göss”) were resistant to antisera against either of the two, whereas strain “Pneum. Do.” was sensitive to antisera against “Pneum. Franz” but not to that against “Pneum. I” (Fig. 2). Mice were pretreated with a constant amount of patient serum taken several days after crisis, then challenged with different pneumococcal strains in serial dilutions, and their time of death recorded.

Fig. 2.

Original table of experiment IV, reproduced from reference [7]. Upper panel Examination of various pneumococcal strains for sensitivity to a donkey antiserum against Pneum. I. Mice were pretreated with 0.2 ml of the antiserum and 3 h later challenged with ten-fold serial dilutions of the pneumococcal cultures (left column). While the mice survive (leben) challenge with Pneum. I, mice die (†) within 1–4 days after challenge with the heterologous strains. Middle panel Same experiment as in upper panel but using a rabbit antiserum against Pneum. Franz. Mice challenged with Pneum. Franz and Pneum. Do. are protected, no protection seen with the other strains. Lower panel Controls without antiserum, all pneumococcal strains kill the mice even at three orders of magnitude lower doses

Controls in this exceptionally detailed and careful study included human sera from healthy individuals or from patients with other diseases, as well as various animal sera (rabbit, donkey, horse) of their own production or provided by others. Notably, a commercial serum was used as well, produced by the company Merck in collaboration with an ophthalmologist named Römer (commonly referred to as the “Römer serum”), and claimed to be polyvalent, i.e. consisting of a pool of several animal species immunized with several pneumococcal isolates [45]. In Neufeld’s and Haendel`s hands, it protected only against Pneum. 1 [6, 7]. The authors conclude: “As brought out by the aforementioned deliberations, the problems posed by a putative serum therapy of pneumococcal infections appear as considerably more complicated than hitherto assumed” [7].

As mentioned above, these results attracted the attention of Simon Flexner who asked Neufeld to send desiccated samples of his pneumococcal strains. While the first shipment in January 1910 was lost (Fig. 3a), the second arrived safely in February (Fig. 3b) [29], and Dochez used it to produce the first so-called univalent (i.e. Type I-specific) antiserum at RIMR. He reported this antiserum protected mice against challenge with about 1/3 of pneumococcal isolates [46]. Neufeld himself seems to have felt that he had done his share, apparently thinking that the application of his findings to the treatment of human pneumonia were in good hands elsewhere, notably at RIMR. Except for two brief reports at meetings of the Berlin Microbiological Society, dealing with attempts to establish a guinea pig model for pneumococcal pneumonia [47, 48],7 studies on human pneumonia were discontinued. Instead, he and Haendel published a final paper summarizing their conclusions, in which they elaborated on three recommendations for clinical trials on pneumococcal serum therapy: “…to decide the question whether a specific therapy of pneumonia presently offers any expectation of success, a series of experiments (should be performed) in which, first, pneumonia patients are injected intravenously with large quantities of serum, second, samples of the serum are exactly calibrated in animal experiments and third, to evaluate the success of the treated cases isolated pneumococci (from each case) get examined for whether they are homologous or heterologous to the serum” [49].

Fig. 3.

a Short note from Flexner alerting Neufeld that the bacteria did not arrive, signature by some aide, not Flexner. b A month later shipment was successful. Letters found among Neufeld’s uncataloged papers at RKI [29]

Although serum therapy of human pneumonia no longer played a prominent role in Neufeld’s own research, he closely observed the international progress in this field. While scattered attempts at the treatment of human pneumonia (or ulcus serpens, an ophthalmological disorder caused by pneumococci) with antisera had been reported since 1891 [3, 50–54], the first comprehensive and systematic analysis, including Type-specific therapy, was published in 1917 by Rufus Cole, Avery, and collaborators at RIMR [14]. Podolsky, in his book Pneumonia before Antibiotics [8], gives an excellent and thorough review of the implementation of antiserum therapy in the USA from 1918 to 1937/1938. This was an effort in both the public and private sectors. Rufus Cole, the Director of the RIMR Hospital, was a staunch advocate for antiserum therapy. Neufeld reviewed this work for a German-speaking audience first in 1922 [55], then again in 1938 [56].8 The 1938 review covered the studies at RIMR as well as subsequent clinical trials in several additional centres in the UK and the US (references in 56), but none in Germany. Neufeld complained: “According to numerous reports that reached us from England and America, hundreds of sick humans had undoubtedly been rescued by intravenous injection of highly active sera against Types I and II, and I have always regretted that nobody here could make up his mind to follow the example from abroad” [23]. But, anyway, time marches on. As Neufeld himself noted [57], by 1938, the sulfonamide drugs became the preferred treatment for pneumococcal pneumonia, and serum therapy became obsolete.

Neufeld became actively involved in research on pneumococcal serotypes only once more, namely when he reproduced Griffith’s transformation experiment in 1928 [58]. This was done together with Walter Levinthal, who had learned to “clone” bacteria from single cells: “We used the one-cell cultures to reproduce the important experiments of F. Griffith on the transformation of avirulent pneumococci (R-forms) into virulent (S-forms) of the same but also of a foreign type… The communication of Griffith just appeared in the Journal of Hygiene (1928, Vol. 27. p. 113); owing to the kindness of Dr. Griffith, who communicated his results to one of us during a visit …in London, we were enabled to begin with our experiments before (Griffith’s paper appeared)” [58]. Because of the “one-cell cultures”, their experiments represented an even more rigorous proof of transformation, because the use of cloned bacteria formally excluded the possibility of contamination. In the Fourth Memorial Griffith Lecture by A. W. Downie, he states that it was Neufeld who visited Griffith at the time Griffith had performed the transformation experiment. Downie notes that Griffith took no further efforts in regard to transformation. In the 1930s, he was very much concerned with the classification of hemolytic streptococci, a major cause of pharyngitis and suppurative complications, and rheumatic fever [19].9

Apparently proud of his own groundbreaking findings, Neufeld never failed to mention the old studies with Haendel in his reviews on pneumococcal serotherapy. In an unpublished draft of around 1930 in the files of his papers at RKI, Neufeld reflected on their consequences: “Although typing research first arose from practical needs, the resulting deep insights into the chemical structure of microorganisms are of far greater general importance….I like to point out that two discoveries of far reaching relevance have arisen from typing research; the discovery of carbohydrates as widely occurring antigenic factors by Avery and Heidelberger and the discovery of the willful change [bold type added] of one pneumococcal type into another by F. Griffith” [29]. Neufeld seems to have anticipated that something fundamental would develop out of Griffith’s observation, but it is likely he did not learn about the elucidation of the transforming principle as DNA, even though it was published a year before he died.

Neufeld’s desideratum

Certain scientists, Neufeld among them, do not get the credit they deserve, in spite of solid and important contributions. Instead, credit often goes to scientists that have a talent to raise public attention for sometimes even mediocre and inconsequential work. A further example of Neufeld’s lack of appreciation is related to the term “opsonin” (from the Greek “οπσονειν”, to prepare for a meal) which was introduced by the British physician Almroth Wright to describe phagocytosis-inducing activities in human blood [59, 60]. This term is still in use today, unlike Neufeld’s and Rimpau’s “bacteriotropin”, describing the exact same phenomenon, only based on much more solid experimental work [40, 41].

Wright had introduced a vaccination therapy for diseases such as tuberculosis and various streptococcal and staphylococcal infections, involving serial injections of heat-killed bacteria [59, 60]. The therapy required tedious monitoring by determination of an “opsonic index”, and Wright’s laboratory was full of trainees from all over the world learning the technique. Wright received much international attention both in the scientific communities and in the public. In a book chapter of 1910 entitled “Opsonins and Vaccine Therapy” and coauthored by Avery, Wright’s work was described in great detail while Neufeld was just mentioned in passing: “If a difference (between normal and immune sera) can be demonstrated, then support would be lent to the theoretical unity of the immune opsonins and ‘bacteriotropins’ as claimed by Neufeld and others” [61]. Even the British playwright George Bernhard Shaw visited Wright’s laboratory and got inspired there for his play “The Doctor’s Dilemma” [23, 42].

Neufeld had little regard for Wright and his work: “We will not go any further into the studies of Wright…. Whether or not the ‘opsonic’ sera have immunizing activity has not been examined by Wright” [40]. But again, his warnings went unheard. In his memoir he writes: “….Wright and coworkers claimed to have surprising success in curing various infections, particularly tuberculosis and staphylococcal diseases. ….Wright’s opsonin diagnosis as well as vaccine therapy were soon forgotten. This is a warning example for how dubious it is to introduce such measures in large scale, whilst it would have been very easy to examine them aforehand in animal experiments” [23].

Neufeld’s publication list comprises studies on a number of additional subjects not mentioned in this paper, including typhoid, tuberculosis, influenza, public health matters, etc [22]. As usual in science, much of this work had addressed then timely issues that have now long been solved. Of lasting interest were his work on disinfectants, intensively discussed by Mozew [22], and his contributions to novel concepts of epidemiology, which have been discussed in depth by Mendelsohn (33; see also footnote 5). Most outstanding, however, were Neufeld’s pioneering studies on pneumococcal serotypes, first exploited in 1916 by Spencer Lister in South Africa for experimenting with a first multivalent pneumococcal vaccine [62]. Human pneumococcal pneumonia is still a pressing problem especially, but not only, in children in the developing world and vaccination strategies continue to rank among today’s challenging research subjects [63–66]. Thus, Neufeld’s contributions are still relevant today, and we firmly believe that they belong in the textbooks in microbiology, immunology, and infectious diseases. With this article, we hope to have given Fred Neufeld’s work its well-deserved recognition.

Note added in proof

After the submission of this paper we took note of the following new publication: Wyres KL et al (2013) Pneumococcal capsular switching: a historical perspective. J Inf Dis 207:439–449. The authors demonstrate that transformation of pneumococcal types occurs naturally and is presumably an immune evasion mechanism selected for in evolution.