Cell. Mol. Life Sci.

(2013) 70:2225–2236
DOI 10.1007/s00018-013-1351-z Cellular and Molecular Life Sciences
MEMORIES AND RETROSPECTIVES

Fred Neufeld and pneumococcal serotypes: foundations

for the discovery of the transforming principle
Klaus Eichmann · Richard M. Krause 

Received: 7 February 2013 / Revised: 23 March 2013 / Accepted: 22 April 2013 / Published online: 21 May 2013
© Springer Basel 2013

Abstract  During the first decade of the twentieth cen- Keywords  History of infectious diseases · History
tury, the German bacteriologist Fred Neufeld, later Direc- of bacteriology · Bile solubility · Quellung reaction ·
tor of the Robert Koch-Institute in Berlin, first described Bacteriotropin
the differentiation of pneumococci into serotypes on the
basis of type-specific antisera. This finding was essen-
tial for subsequent research at the Rockefeller Institute of Introduction
Medical Research (RIMR) in New York, and elsewhere,
aiming for the conquest of human pneumococcal pneu- Ever since Emil von Behring and Shibasaburo Kitasato had
monia, including antiserum therapy, the discovery that the reported in 1890 that infected animals can be cured by anti-
type-specific antigens were carbohydrates, and the devel- serum of animals immunized with the corresponding bacte-
opment of effective multivalent pneumococcal polysac- rial toxins [1, 2], attempts were made worldwide to combat
charide vaccines. Moreover, on the basis of pneumococcal infectious diseases with immune sera. Two physicians, the
serotypes Fred Griffith, in 1928 in London, discovered brothers Felix and Georg Klemperer, observed as early as
pneumococcal transformation, and Oswald T. Avery and 1891 that animals inoculated with the sputum of pneumo-
coworkers, in 1944 at RIMR, identified DNA as the trans- nia patients became immune, and that patient sera could
forming substance. This sequence of events, leading to protect animals against subsequent infection with “Fraen-
today’s knowledge that genes consist of DNA, was initi- kel’s diplococcus” [3]. Later dubbed streptococcus pneu-
ated by a farsighted move of Simon Flexner, first Director moniae, or pneumococcus, these bacteria had first been
of the RIMR, who asked Neufeld to send his pneumococ- described 1886 by Albert Fraenkel as a cause of human
cal typing strains, thus setting the stage for pneumococ- pneumonia [4]. The Klemperers’ observations stimulated a
cal research at RIMR. Here, we describe Fred Neufeld’s great number of physicians and microbiologists, to engage
contributions in this development, which have remained in studies to develop a serum therapy for human pneumo-
largely unknown. nia, then a leading cause of death. One of them was Fred
Neufeld, at the time employed at the Robert Koch Insti-
tute 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
K. Eichmann (*)  detail below, Neufeld, together with a colleague, Staabsarzt
Max Planck Institute of Immunobiology and Epigenetics,
Dr. Haendel, were the first to discover pneumococcal sero-
Stübeweg 51, 79108 Freiburg, Germany
e-mail: eichmann@immunbio.mpg.de types, and to observe that protection by immune serum was
type-specific. They published this in several papers during
R. M. Krause  1909 [5–7].
National Institute of Allergy and Infectious Diseases,
Since its foundation in 1910, physician-scientists at
National Institutes of Health, 16 Center Drive, Bethesda,
MD 20892, USA the Rockefeller Institute’s Hospital in New York have
e-mail: Richard_Krause@nih.gov been engaged in studying the pressing problem of human

13
2226 K. Eichmann, R. M. Krause

pneumococcal pneumonia [8]. Simon Flexner, the first Transforming Principle [17] remarks: “It is often pointed
Director of RIMR, took note of Neufeld’s work on pneu- out that research in the basic sciences provides the base of
mococcal serotypes and, immediately realizing its impor- new knowledge essential for the development of the applied
tance, asked Neufeld to provide samples. Flexner urged sciences, including medicine. We are less frequently
Rufus Cole, Director of the Rockefeller Hospital, to hire reminded that the reverse can also occur. From the initial
Oswald T. Avery, then working at the Hoagland Laboratory discovery of the phenomenon known as ‘the transformation
under the direction of Benjamin White, a chemist by train- of pneumococcal types’ until the identification of the trans-
ing. This experience prepared Avery in subsequent years to forming substance as DNA, all of the researchers were
determine, with his colleague Michael Heidelberger, the medical bacteriologists primarily interested in the cause
pneumococcal antigens that induced protective immunity and control of human pneumonia” [17]. The history and the
as carbohydrates [9, 10] and, together with Colin MacLeod individuals involved have been extensively and thoroughly
and Maclyn McCarty, the chemical nature of the transform- covered in the literature, notably in two books, one by
ing principle as DNA [11]. Maclyn McCarty [17], the other by René Dubos [18], as
Avery arrived at RIMR in 1913 and joined forces with well as elsewhere [19, 20]. We will make no attempt to
Alphonse R. Dochez who had already began to study repeat this saga here, and only mention the highlights.2
human immunity to pneumococci [12]. In 1915, Avery Briefly, Avery and collaborators were intrigued by an
and Dochez reported initial studies of pneumococcal types experiment published in 1928 by Fred Griffith in the UK,
and their relationship to human pneumonia [13], followed in which Griffith showed that avirulent pneumococci could
in 1917 by the Rockefeller Institute Monograph No. 7 in be transformed into virulent ones by coinoculation of mice
which Avery, Chickering, Cole, and Dochez presented a with strains of both kinds [21]. Avirulent pneumococci,
comprehensive and authoritative account of success in also referred to as R-forms because they grow as rough
treating pneumonia with type-specific horse antiserum, an colonies on agar, arise during culture passages due to loss
astonishing feat at the time [14]. of the carbohydrate capsule. They are non-pathogenic
It is no surprise that this report led to many efforts par- because they are sensitive to enzymatic attack in the host.
ticularly in the USA to produce horse antisera and their use Virulent pneumococci, owing to their capsule, are protected
to treat pneumococcal pneumonia. The textbooks of medi- from enzymatic attack and cause septicemia in the mouse.
cine of that era as well as the first and second editions of They are referred to as S-forms because they form smooth
Principles of Bacteriology and Immunity by Topley and colonies.
Wilson noted reports of many clinical trials with mixed Griffith reported that upon co-inoculation of a mouse
results [15]. Far too frequently, clinical trials reported little with a live R-strain and an excess of heat-killed S-bacteria,
or no success with antisera therapy. Type I and Type II anti-
sera were often successful but not Type IV and other types. 2
  Both authors of this article have been members of the scientific staff
Later, it was learned that the antibodies in certain horse of the Rockefeller University, formerly named Rockefeller Institute
antisera lacked phagocytic properties which was responsi- for Medical Research (RIMR), New York: R.M.K. having spent a
substantial part of his professional life there, K.E. several years as a
ble for failure of antiserum therapy. Yet it is no surprise that junior scientist. Neither of the authors had the good fortune to know
there were hundreds of reports on efforts to produce effec- Avery while he was still an active investigator. R.M.K. met Avery in
tive antisera for therapy. The stakes were high. Pneumonia 1950 at a conference on streptococcal research at the Streptococcal
was one of the most common causes of death in that era. Diseases Laboratory, Warren Air Force Base, Cheyenne, Wyoming.
He reviewed his research on M protein isolation, a virulence factor
After Michael Heidelberger and Avery reported that the of Group A streptococci, with both Avery and Rebecca Lancefield.
type-specific pneumococcal antigens were polysaccharides, Avery’s advice was: “Treat M protein more gently.” Avery retired
there were many efforts to develop a pneumococcal vaccine, from the RIMR and from active science in 1948. MacLeod left Rock-
all ending in failure. Success was not achieved until a clini- efeller in 1941 to become professor of microbiology at New York Uni-
versity School of Medicine. McCarty remained at Rockefeller Uni-
cal trial at a US Army military base in 1944 [16]. But for versity for many years until his death in 2005. He was active during
many technical practical reasons, the current highly success- retirement in research, editing the Journal of Experimental Medicine,
ful pneumococcal vaccine was not developed until the 1970s. and many related activities. Both of the authors had the good fortune
The experiments that led to the discovery of the trans- to work in close association with McCarty—Mac as he was known
to all his associates—whose laboratory and office were next door to
forming principle had originally been done with the aim to ours. He was our respected teacher and advisor in many aspects of
further unravel pneumococcal pathogenicity. McCarty in science and everyday life. It is perhaps not surprising that, as a result,
the chapter “The Sugarcoated Microbe”,1 in his book The both of us developed a deep and lasting interest in the concepts and
experiments that eventually led to the identification of DNA as the
transforming principle. This discovery, published in 1944, became the
1
  The phrase “the sugarcoated microbe” has been envisaged by Avery basis of today’s knowledge that genes consist of DNA, and is perhaps
as the title of a book he wanted to write but never did. It was adopted the most consequential discovery in the biosciences of the twentieth
by McCarty as a chapter title [17]. century.

13
Fred Neufeld: from pneumococcal serotypes to DNA 2227

live virulent S-bacteria can be recovered from the mouse was moderately well to do, Neufeld could study medicine
that produce a capsule and maintain virulence for many and passed his final examination in Heidelberg in 1892. A
generations. Thus, avirulent non-encapsulated cocci had year later, he obtained his doctoral degree with the grade
been transformed into virulent encapsulated ones. Moreo- “insigni cum laude” (second best) with a written thesis on
ver, if the coinoculated pneumococcal strains differed in “Case Studies on Innate Tumors of the Scull” [22].
serotype, the recovered live bacteria expressed the serotype After a year of work as a physician, in 1894 Neufeld
of the heat-killed S-strain, not their own original serotype. joined the Royal Prussian Institute of Infectious Diseases in
Griffith concluded that the live avirulent cocci had engulfed Berlin as a deputy assistant without pay. This institute, in
material from the heat-killed virulent bacteria, a process 1912 renamed Royal Institute of Infectious Diseases Rob-
that enabled them to express a novel carbohydrate capsule. ert Koch, is commonly referred to as the Robert Koch Insti-
He envisaged this material as “pabulum”, something to tute (RKI). In 1897, Neufeld was promoted to staff assist-
eat [21]. ent, meaning that he received a salary. His initial
The objective of Avery and co-workers was to identify assignment was to work on cholera, but in his memoir he
the chemical nature of the pabulum. To this end, they first stated that “this plague was already close to having van-
developed an in vitro system that allowed them to transform ished at the time, (so that) I was searching for new fields of
avirulent cocci in a test tube, using extracts from virulent activity” [23].3 As a result, Neufeld turned to the study of
bacteria. They then fractionated the extract, using various typhoid, and in his first publication he proudly reported the
methods available at the time. Depletion of proteins, lipids, detection of typhoid bacilli in tissue recovered from roseola
or carbohydrates did not affect transforming activity. In con- lesions [24], raising some interest in the medical commu-
trast, a highly purified fraction that contained nucleic acids nity [22, 23].
retained transforming potential. DNAse destroyed it, RNAse As of 1900, Neufeld became enrolled in Koch’s stud-
did not. They thus showed that Type II R-forms could be ies on malaria and tuberculosis, and in 1903 he accompa-
transformed into Type III S-forms using DNA from Type III nied Koch to Rhodesia to investigate East Coast Fever of
pneumococci [11]. The results, exciting and far reaching as cattle. He did not seem to have overly enjoyed the tropi-
they turned out to be, were not directly relevant to the con- cal experience: “Beginning of 1904 the work on the new
quest of pneumonia, the objective of the Avery laboratory. disease had progressed sufficiently so that I could quit
McCarty remarked: “Admittedly, in the later stages of the and return to Berlin to continue working on tuberculosis.
search we came to see that our findings would not help to On the side, I could concentrate on my serological inter-
eradicate pneumonia” [17]. ests” [23]. By now having been promoted to Professor, in
Pneumococcal serum therapy, the transformation of 1904 Neufeld was commissioned as advisor to the Imperial
pneumococcal types, the development of pneumococcal Health Service in Berlin, and became fully employed there
vaccines, and the discovery of the transforming principle— as a research officer from 1907 to 1912. Neufeld returned
all four rested heavily on Neufeld’s original discovery of to the RKI in 1912, now as department head. When the post
pneumococcal serotypes. Although Neufeld’s work is to of director became vacant, Neufeld was offered the direc-
some extent quoted in the relevant literature, his name and torship, but only after three other candidates had declined.
contributions are mentioned only briefly and in passing [8, On Sept 1, 1917, Neufeld became Director of the RKI [22,
17–19]. As a result, we felt that the groundbreaking role of 25–27].
Neufeld’s contributions as foundations of the subsequent When Neufeld became director, the Institute had six
developments has not been adequately appreciated. departments, with one of them, Bacteriology, led by Neu-
When we took a closer look at Fred Neufeld, we feld himself. Under Neufeld’s leadership the RKI expanded
detected not only a creative and farsighted scientist but also to 10 departments under varying names such as: Plagues,
a man of upright character who combined professional and Tropical Diseases, Rabies, Chemistry, Medical Chemistry,
private integrity in difficult times. Serology, Smallpox, Chemotherapy, etc. After WWI and
during the Weimar Republic, it became more and more dif-
ficult to secure sufficient resources for research. Important
Education and career work was still done, but less than before WWI. An Emer-
gency Association (Notgemeinschaft) of German Science
Fred Julius Neufeld was born on February 17, 1869 in was established that provided research grants on a competi-
Neuteich, a small village near Danzig, West Prussia, (now tive basis. By far the largest individual grant was given to
Novy Staw, Poland). He was the only son of Dr. Hermann Neufeld for studies on BCG vaccination. Several RKI sci-
Neufeld, a general practitioner, and his wife Johanna. The entists including Neufeld were supported by grants from
family was part of a Mennonite community that had settled
in West Prussia in the sixteenth century. Because the family 3
  All quotations from documents in German translated by K. E.

13
2228 K. Eichmann, R. M. Krause

the Rockefeller Foundation, which kept an office in Paris Neufeld’s Harvey Lecture, delivered in New York on
for European applications. Efforts were made to maintain October 30, 1926, was entitled “Origin and Dissemination
international relations: Prominent scientists from all over of Tuberculosis According to Recent Investigations” [31].
the world visited the RKI during these years, including Interestingly, in this and other lectures, Neufeld often criti-
Simon Flexner, Rufus Cole and Theobald Smith, all from cally reviewed the work of others, with minimal reference
RIMR. The role of the RKI as an educational center for for- to his own contributions. Here, he referred to his former
eign visiting fellows resumed after WWI by attracting par- mentor Robert Koch who, in the ongoing controversy about
ticularly Japanese and Russian guest scientists. Although the relationships of bovine and human tuberculosis, had
German scientists were still discriminated against at inter- proposed “…the view, held before him by Theobald Smith,
national conferences and commitees, Neufeld participated that the infective agents of bovine and human tuberculosis
in the international serum standardization program of the are different from each other”. Neufeld felt obliged to men-
League of Nations and attended the serum conferences in tion “…the quite justifyable opposition which this statement
Paris 1922 and Geneva 1924 [22, 25–28]. in his London Lecture (of 1901) had aroused…”, not with-
Of far reaching consequence was the relationship of out stressing that Koch would have been more convincing
Neufeld and Simon Flexner, first director of the RIMR in had “…a clear differentiation between bovine and human
New York. The relationship may have started in 1910 when tuberculosis…. been established and the means indicated
Flexner asked Neufeld to provide samples of the pneumo- whereby both types of bacilli could be distinguished…”
coccal serotypes that Neufeld had identified. A highlight of [31]. We now know that, even without this evidence, Koch
their friendship was Flexner’s invitation to Neufeld, in 1925, was right.
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 Nazi rule, resignation, and retirement
Harvey Lecture—an outstanding honour—and several other
prestigious lectures at major US universities. Flexner returned In early 1933, at age 64, Neufeld fell ill with what was
Neufeld’s visit in 1927, an event that was covered by the New referred to in his physician’s statement as a “severe influ-
York Times and major German newspapers [29, 30] (Fig. 1). enza” (schwere Grippe) leading to a “considerable

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

13
Fred Neufeld: from pneumococcal serotypes to DNA 2229

general state of exhaustion which will take several months that his illness was the only reason and that he had no dif-
to resolve” [29]. On March 1, Neufeld asked the Minister ficulties with the authorities: “My retirement was voluntary
of the Interior for 3 months sick leave, and, on May 23, and (as I have a clear Aryan pedigree) the only reason for
he wrote a letter of resignation. The reasons for this move it—and a sufficient reason it seems to me—was my disease.
have never been unequivocally determined, and have been Otherwise, the government would not have made me a hon-
the subject of much speculation. Here, we briefly summa- orary member of the Institute” [25, 29].
rise the circumstances preceding Neufeld’s resignation, for Nevertheless, there are reasons to assume that the Nazi
more detail the reader is referred to references [22, 25, 26], government was not altogether unhappy with Neufeld’s
and further references cited therein. early retirement [22, 25, 26]. Neufeld had never joined the
The Nazis seized power in Germany on January 30, Nazi party, although everyone carrying official responsi-
1933. One of the first laws the Nazis passed was the bilities in Germany was strongly encouraged to do so.
“Gesetz zur Wiederherstellung des Berufsbeamtentums” Moreover, Neufeld was opposed to the prevailing attitude,
(Law to reestablish the professional public service), which favoured by the Nazis, that all microorganisms should be
in fact was a law to discharge Jews from state employment. indiscriminately eradicated by all available means, and
As a result, seven employed Jewish research scientists of strongly criticized the exaggerated procedures of quaran-
the RKI were handed their dismissal documents on March tine and disinfection then in use [32]. This was very much
31, 1933, to become effective by June 30. A few days later, in contrast to his successor, Kleine, who was Hermann
the date was changed to May 15, indicating how urgent Göring’s favorite because of a “more appropriate, mili-
the matter had to be handled. In addition to the seven staff tary, colonial, and microbe-hunting career” [33].5 In any
scientists, at least three Jewish technical assistants and at case, in his letter of acceptance the Prussion Minister of
least five voluntary (unpayed) Jewish junior scientists had the Interior, Göring, thanked Neufeld profusely for his
to leave. engagement and informed him that he would be allowed
Neufeld as director of the RKI had hired most of the to continue working at the RKI as a honorary member,
senior scientists during the pre-Nazi period, in spite of the and to use his laboratories and the “required facilities at
anti-Semitism already prevalent at that time. They repre- no cost” [29]. The Rockefeller Foundation resumed sup-
sented the promising talent of the RKI, such as Fritz Kauff- porting his work and Neufeld could continue his studies,
mann, whom Neufeld had recommended to Flexner for a but now without responsibilities for the RKI’s new
visit to the RIMR, and who was to become known for the policies.
Kauffmann–White scheme for serotyping salmonella The correspondence between Neufeld and Flexner con-
strains. Moreover, internationally respected scientists tinued until 1935, and there are no letters between them
including Walter Levinthal,4 who had received the Paul after that [29, 30]. Noted here are excerpts of an exchange
Ehrlich Prize, and who had worked at RIMR, Alfred Cohn, of letters in 1935. Neufeld and Flexner had now retired as
later professor at Yale, and Neufelds’s favorite coworker, Directors.
Rochla Etinger–Tulczynska, were among those who had to Letter from Neufeld to Flexner, August 3, 1935:
leave. (For a full account of the names and their further
“The Koch Institut is now being merged with the bac-
fate, see [25].)
teriological division of the Reichsgesundheitsamt –
Neufeld learned of the discharge of his scientific staff
which I do not consider fortunate….
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
5
that had discharged most of its leading scientific ranks. Sig-  This aspect was analysed in depth by Mendelson [33]: The
“microbe-hunters” among microbiologist followed Robert Koch
nificantly, he applied to retire as of July 1, the very date of who concluded from his successful handling of the cholera epidemic
dismissal of his colleagues [25, 26, 29]. of Hamburg in 1892: “Had one in Hamburg not tracked the cholera
In the US and England, it was suspected that Neufeld’s into its remotest nook and cranny in so energetic a way and rendered
resignation was not entirely voluntary but forced by the innocious every discoverable trace of the infectious material, then it
certainly would not have been possible, in my conviction, to master
Nazis. In several letters to people that offered to accom- the fuel that was spread over the city in such a massive way”. In con-
modate him if he were to leave Germany, Neufeld insisted trast, Neufeld attacked this attitude by calling upon his colleagues:
“…to give up at last the notion that it is their duty to track down every
last bacillus (or in typhus every last louse) into its remotest nook and
cranny and to kill it” [32]. Neufeld thus held what became known as
4
 Levinthal moved to England in 1933, and became professor of the “holistic view”, i.e. the coexistence of man and microbes in some
microbiology at Edinborough. His correspondence and papers were form of equilibrium, with epidemics being mere disturbances of that
discarded after his death. equilibrium. See also reference [22].

13
2230 K. Eichmann, R. M. Krause

I had the luck of having, – in Levinthal, Frau Etinger- of a specific serum single microorganisms get clumped
Tulczynska and recently in Sevag6 who was born in together in a big lump, then an agglutination in this
Armenia but is an American citizen, – distinguished, sense….has so far never been described”. What rather hap-
original and gifted collaborators”. pens is this: “Upon mixing of equal parts of agglutinating
serum and pneumococcal broth culture,…..one observes
Letter from Flexner to Neufeld September 5, 1935:
in a hanging drop a distinct swelling (Quellung) phenom-
“It is sad to think of the Koch Institute losing its iden- enon that, with a strong acting serum, occurs immediately
tity. It can never lose its place as the foremost institu- or within a few minutes” [36]. The phenomenon became
tion of bacteriology in history, and some day it may internationally known under the German term “Quellung
come back again to renew its youth and purpose. I Reaction”. To identify serotypes of pneumococci isolated
sincerely hope it may.….Levinthal seems to be doing from patients who received homologous serum therapy,
well in London. I am very glad”. Avery et al. [14] used a more rapid variant of the Quellung
Reaction by testing peritoneal fluid of mice inoculated 4 h
What is clear in a review of these letters from 1910 to
earlier with the patients’ sputum. To further speed up the
1935 [29, 30] is that both men had great respect and affec-
selection of the proper serum for treatment, British inves-
tion for each other.
tigators reported in 1932 that patient sputum could be used
Neufeld was active in the laboraory until at least 1943
directly for pneumococcal typing by the Quellung reaction
when he still published two papers. In 1944, at his 75th
[37, 38]. Only then did Neufeld consider this reaction for
birthday, Neufeld was awarded the Goethe Medal, then the
rapid bacteriologic diagnosis [39].
highest honour in arts and sciences in Germany. Neufeld
Neufeld was more interested in developing a theory of
died on April 18, 1945, during the final battle of WWII in
agglutination on the basis of the Quellung phenomenon
Berlin. The obituary, written by Kleine, gave as the cause
[36], a rather elegant one considering how little was known
of death extreme fatigue and exhaustion [34].
about antibodies. In the following papers, he and Walter
Rimpau demonstrated that the immune defence against
pneumococci (and streptococci) involved the combined
Pneumocccal studies prior to serotypes
action of antibodies (they suggested the term “bacteriot-
ropins”) and phagocytes, but did not require complement
Neufeld began to work with pneumococci during his first
[40, 41]. On that basis, they developed “the bacteriotropin
period at the RKI when he had not yet decided to concen-
theory” [41], which in fact represented the first unify-
trate on one of several microbial infections under study
ing concept of the coordinated function of antibodies and
there, including cholera, typhoid, tuberculosis, among
phagocytes in anti-bacterial immunity. Neufeld and Rim-
others. But already in his first papers on pneumococci, he
pau thus felt that they had resolved the Ehrlich/Metchnikov
described two observations that were to become, albeit
conflict, for decades the dominating controversy in immu-
without Neufeld suggesting so, the basis of widely used
nology on the roles of antibodies and phagocytes in immu-
diagnostic tests: the bile solubility of pneumococci and the
nity, i.e. between “humoral” and “cellular” immunity ([42],
Quellung reaction. His paper on bile solubility appeared in
with further references therein). Although essentially cor-
1900, and described the specific lytic effect for pneumo-
rect, Neufeld’s and Rimpau’s ideas could not compete with
cocci by the bile of several animals [35]. This lytic effect
those of the science giants Ehrlich and Metchnikov and
of bile became a standard bacteriologic test to distinguish
their respective followers, and so the struggle went on for
pneumococci from streptococci. The latter were resistant to
years to come. Neufeld states in his memoir: “It is amaz-
the lytic effect of bile.
ing for how long the controversy went on between the
Of still greater diagnostic importance was the Quellung
‘humoral’ conception of immunity, maintained in Germany
phenomenon, first published by Neufeld in 1902, in a paper
under the impression of the discoveries of Behring, Pfeiffer,
dealing with bacterial agglutination. Neufeld writes: “In
and Ehrlich, and the ‘phagocyte theory’, fostered in France
all of these investigations Fraenkel’s pneumococci have
by Metchnikov and his numerous disciples at Pasteur” [23].
received little attention. Indeed, if one understands agglu-
tination as usual, i.e. as a process in which upon addition
Pneumococcal serotypes
6
  M. G. Sevag described a method of deproteinizing solutions which In many of his papers [5, 36, 40, 41, 43], Neufeld described
was used by Avery et al. in the purification of the DNA fraction that
had transforming activity (quoted in [11]). Neufeld was very support-
in much detail his methods to produce and test pneumo-
ive of Sevag, for example by approaching Flexner to consider one of coccal antisera. He was not the only one to try to generate
Sevag’s papers for the Journal of Experimental Medicine. potent sera for therapeutic purposes, and opinions differed

13
Fred Neufeld: from pneumococcal serotypes to DNA 2231

Fig. 2  Original table of experiment IV, reproduced from reference within 1–4 days after challenge with the heterologous strains. Middle
[7]. Upper panel Examination of various pneumococcal strains for panel Same experiment as in upper panel but using a rabbit antise-
sensitivity to a donkey antiserum against Pneum. I. Mice were pre- rum against Pneum. Franz. Mice challenged with Pneum. Franz and
treated with 0.2 ml of the antiserum and 3 h later challenged with Pneum. Do. are protected, no protection seen with the other strains.
ten-fold serial dilutions of the pneumococcal cultures (left column). Lower panel Controls without antiserum, all pneumococcal strains
While the mice survive (leben) challenge with Pneum. I, mice die (†) kill the mice even at three orders of magnitude lower doses

widely about techniques and protocols. Two questions were isolate protected against all pneumococcal strains, it was
of particular interest, the method of testing the antisera, also proposed that an antiserum would protect only against
and that of their “valency”. As to the test methods, Neu- the strain used for immunization [44, 45]. After discuss-
feld kept stressing the quantitative aspect, i.e. the need to ing the situation, Neufeld and Haendel described their
carefully calibrate a serum by titrating it against a series of own observations: “Our experiments yielded the follow-
dilutions of the bacteria, in a mouse protection assay. Quite ing: Using a univalent highly active serum against a sin-
frequently, he criticized his competitors, if they had pub- gle strain, as well as several patients’ convalescent sera,
lished results at variance with his own, for using inadequate we observed protection not only against the homologous
analytical techniques. strain, but also against a series of other strains, and the
Opinions on valency also varied widely. While most protection was quantitatively not significantly lower. ….
experts held the view that an antiserum against a single After we had obtained similar results with a series of

13
2232 K. Eichmann, R. M. Krause

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]

cultures, we were surprised to see no effect of our serum on In a continuation of this study, Neufeld and Haendel
cultures isolated from two cases of pneumonia that clini- identified three pneumococcal serotypes [6, 7]. Here, we
cally presented nothing unusual….Also there was no effect reproduce only one of a series of experiments in which
of our human convalescent sera”. They considered the they showed that an antiserum against the prototype strain,
existence of “serum-fast” pneumococci, but rejected the termed “Pneum. I”, did not protect against strain “Pneum.
possibility because they found that the post-crisis serum of Franz”, named after the patient from which it was isolated,
one of the patients protected mice against pneumococci of and vice versa. Moreover, pneumococci from two other
both atypical isolates [5]. patients (“Pneum. Br.”, “Pneum. Göss”) were resistant to
The latter finding was the starting point for a compre- antisera against either of the two, whereas strain “Pneum.
hensive clinical study on the role of antibodies in the recov- Do.” was sensitive to antisera against “Pneum. Franz” but
ery of human patients from pneumonia, as manifested in not to that against “Pneum. I” (Fig. 2). Mice were pretreated
the dramatic drop of temperature, known as the “crisis”. with a constant amount of patient serum taken several days
The first aim of the study was to prove that sera taken after after crisis, then challenged with different pneumococcal
crisis contained protective antibodies, which was a contro- strains in serial dilutions, and their time of death recorded.
versial issue at the time. Neufeld and Haendel found anti- Controls in this exceptionally detailed and careful study
bodies in every case: “We not only became convinced that included human sera from healthy individuals or from
the pneumonic crisis is exclusively due to antibodies, but patients with other diseases, as well as various animal sera
that these antibodies are also experimentally demonstra- (rabbit, donkey, horse) of their own production or provided
ble….Above all, the antibodies found in the blood of conva- by others. Notably, a commercial serum was used as well,
lescents behave exactly like those artificially generated in produced by the company Merck in collaboration with an
experimental animals….” [6]. ophthalmologist named Römer (commonly referred to as

13
Fred Neufeld: from pneumococcal serotypes to DNA 2233

the “Römer serum”), and claimed to be polyvalent, i.e. implementation of antiserum therapy in the USA from
consisting of a pool of several animal species immunized 1918 to 1937/1938. This was an effort in both the public
with several pneumococcal isolates [45]. In Neufeld’s and and private sectors. Rufus Cole, the Director of the RIMR
Haendel`s hands, it protected only against Pneum. 1 [6, Hospital, was a staunch advocate for antiserum therapy.
7]. The authors conclude: “As brought out by the afore- Neufeld reviewed this work for a German-speaking audi-
mentioned deliberations, the problems posed by a putative ence first in 1922 [55], then again in 1938 [56].8 The 1938
serum therapy of pneumococcal infections appear as con- review covered the studies at RIMR as well as subsequent
siderably more complicated than hitherto assumed” [7]. clinical trials in several additional centres in the UK and
As mentioned above, these results attracted the atten- the US (references in 56), but none in Germany. Neufeld
tion of Simon Flexner who asked Neufeld to send desic- complained: “According to numerous reports that reached
cated samples of his pneumococcal strains. While the first us from England and America, hundreds of sick humans
shipment in January 1910 was lost (Fig. 3a), the second had undoubtedly been rescued by intravenous injection of
arrived safely in February (Fig. 3b) [29], and Dochez used highly active sera against Types I and II, and I have always
it to produce the first so-called univalent (i.e. Type I-spe- regretted that nobody here could make up his mind to fol-
cific) antiserum at RIMR. He reported this antiserum pro- low the example from abroad” [23]. But, anyway, time
tected mice against challenge with about 1/3 of pneumo- marches on. As Neufeld himself noted [57], by 1938, the
coccal isolates [46]. Neufeld himself seems to have felt sulfonamide drugs became the preferred treatment for
that he had done his share, apparently thinking that the pneumococcal pneumonia, and serum therapy became
application of his findings to the treatment of human obsolete.
pneumonia were in good hands elsewhere, notably at Neufeld became actively involved in research on pneu-
RIMR. Except for two brief reports at meetings of the mococcal serotypes only once more, namely when he
Berlin Microbiological Society, dealing with attempts to reproduced Griffith’s transformation experiment in 1928
establish a guinea pig model for pneumococcal pneumo- [58]. This was done together with Walter Levinthal, who
nia [47, 48],7 studies on human pneumonia were discon- had learned to “clone” bacteria from single cells: “We used
tinued. Instead, he and Haendel published a final paper the one-cell cultures to reproduce the important experi-
summarizing their conclusions, in which they elaborated ments of F. Griffith on the transformation of avirulent pneu-
on three recommendations for clinical trials on pneumo- mococci (R-forms) into virulent (S-forms) of the same but
coccal serum therapy: “…to decide the question whether a also of a foreign type… The communication of Griffith just
specific therapy of pneumonia presently offers any expec- appeared in the Journal of Hygiene (1928, Vol. 27. p. 113);
tation of success, a series of experiments (should be per- owing to the kindness of Dr. Griffith, who communicated
formed) in which, first, pneumonia patients are injected his results to one of us during a visit …in London, we were
intravenously with large quantities of serum, second, enabled to begin with our experiments before (Griffith’s
samples of the serum are exactly calibrated in animal paper appeared)” [58]. Because of the “one-cell cultures”,
experiments and third, to evaluate the success of the their experiments represented an even more rigorous proof
treated cases isolated pneumococci (from each case) get of transformation, because the use of cloned bacteria for-
examined for whether they are homologous or heterolo- mally excluded the possibility of contamination. In the
gous to the serum” [49]. Fourth Memorial Griffith Lecture by A. W. Downie, he
Although serum therapy of human pneumonia no longer states that it was Neufeld who visited Griffith at the time
played a prominent role in Neufeld’s own research, he Griffith had performed the transformation experiment.
closely observed the international progress in this field. Downie notes that Griffith took no further efforts in regard
While scattered attempts at the treatment of human pneu- to transformation. In the 1930s, he was very much con-
monia (or ulcus serpens, an ophthalmological disorder cerned with the classification of hemolytic streptococci, a
caused by pneumococci) with antisera had been reported major cause of pharyngitis and suppurative complications,
since 1891 [3, 50–54], the first comprehensive and system- and rheumatic fever [19].9
atic analysis, including Type-specific therapy, was pub- Apparently proud of his own groundbreaking findings,
lished in 1917 by Rufus Cole, Avery, and collaborators at Neufeld never failed to mention the old studies with Haen-
RIMR [14]. Podolsky, in his book Pneumonia before Anti- del in his reviews on pneumococcal serotherapy. In an
biotics [8], gives an excellent and thorough review of the unpublished draft of around 1930 in the files of his papers

8
7
  This animal model was not further pursued. It was replaced by stud-   Both reviews are transcripts of lectures given by Neufeld about a
ies on a spontaneous pneumococcal epidemic of animal house guinea year before publication.
9
pigs, in line with Neufeld's philosophy that studying natural infections   Griffith died in 1941 during an air raid on London. No correspond-
was more informative than artificial animal models [22]. ence between Griffith and Neufeld in the 1930s has been found.

13
2234 K. Eichmann, R. M. Krause

at RKI, Neufeld reflected on their consequences: “Although went unheard. In his memoir he writes: “….Wright and
typing research first arose from practical needs, the result- coworkers claimed to have surprising success in curing var-
ing deep insights into the chemical structure of microor- ious infections, particularly tuberculosis and staphylococ-
ganisms are of far greater general importance….I like to cal diseases. ….Wright’s opsonin diagnosis as well as vac-
point out that two discoveries of far reaching relevance cine therapy were soon forgotten. This is a warning example
have arisen from typing research; the discovery of carbo- for how dubious it is to introduce such measures in large
hydrates as widely occurring antigenic factors by Avery scale, whilst it would have been very easy to examine them
and Heidelberger and the discovery of the willful change aforehand in animal experiments” [23].
[bold type added] of one pneumococcal type into another Neufeld’s publication list comprises studies on a num-
by F. Griffith” [29]. Neufeld seems to have anticipated that ber of additional subjects not mentioned in this paper,
something fundamental would develop out of Griffith’s including typhoid, tuberculosis, influenza, public health
observation, but it is likely he did not learn about the eluci- matters, etc [22]. As usual in science, much of this work
dation of the transforming principle as DNA, even though it had addressed then timely issues that have now long been
was published a year before he died. solved. Of lasting interest were his work on disinfectants,
intensively discussed by Mozew [22], and his contribu-
tions to novel concepts of epidemiology, which have been
Neufeld’s desideratum discussed in depth by Mendelsohn (33; see also footnote
5). Most outstanding, however, were Neufeld’s pioneer-
Certain scientists, Neufeld among them, do not get the ing studies on pneumococcal serotypes, first exploited in
credit they deserve, in spite of solid and important contri- 1916 by Spencer Lister in South Africa for experiment-
butions. Instead, credit often goes to scientists that have a ing with a first multivalent pneumococcal vaccine [62].
talent to raise public attention for sometimes even medio- Human pneumococcal pneumonia is still a pressing prob-
cre and inconsequential work. A further example of Neu- lem especially, but not only, in children in the developing
feld’s lack of appreciation is related to the term “opsonin” world and vaccination strategies continue to rank among
(from the Greek “οπσονειν”, to prepare for a meal) which today’s challenging research subjects [63–66]. Thus, Neu-
was introduced by the British physician Almroth Wright to feld’s contributions are still relevant today, and we firmly
describe phagocytosis-inducing activities in human blood believe that they belong in the textbooks in microbiology,
[59, 60]. This term is still in use today, unlike Neufeld’s immunology, and infectious diseases. With this article, we
and Rimpau’s “bacteriotropin”, describing the exact same hope to have given Fred Neufeld’s work its well-deserved
phenomenon, only based on much more solid experimental recognition.
work [40, 41].
Wright had introduced a vaccination therapy for dis- Note added in proof  After the submission of this paper we
eases such as tuberculosis and various streptococcal and took note of the following new publication: Wyres KL et al
staphylococcal infections, involving serial injections of (2013) Pneumococcal capsular switching: a historical per-
heat-killed bacteria [59, 60]. The therapy required tedious spective. J Inf Dis 207:439–449. The authors demonstrate
monitoring by determination of an “opsonic index”, and that transformation of pneumococcal types occurs naturally
Wright’s laboratory was full of trainees from all over the and is presumably an immune evasion mechanism selected
world learning the technique. Wright received much inter- for in evolution.
national 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 Acknowledgments  The authors are grateful for the assistance of
Ms Heide Tröllmich, Archivist of the Robert Koch Institute for access
work was described in great detail while Neufeld was just by K. E. to the Fred Neufeld files containing extensive papers and
mentioned in passing: “If a difference (between normal and correspondence, and for the assistance of Mr Charles Greifenstein,
immune sera) can be demonstrated, then support would be Archivist of the American Philosophical Society for access by R. M.
lent to the theoretical unity of the immune opsonins and K. to the Simon Flexner files. There is extensive correspondence in
both archives between Simon Flexner and Fred Neufeld. We are grate-
‘bacteriotropins’ as claimed by Neufeld and others” [61]. ful also to Prof. Hans-Hartmut Peter and to Sir Peter Lachmann for
Even the British playwright George Bernhard Shaw visited helpful suggestions and comments.
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 References
will not go any further into the studies of Wright…. Whether
or not the ‘opsonic’ sera have immunizing activity has not 1. Behring E (1890) Untersuchungen über das Zustandekommen der
been examined by Wright” [40]. But again, his warnings Diphterieimmunität bei Thieren. Dtsch Med Wochenschr 16:1145

13
Fred Neufeld: from pneumococcal serotypes to DNA 2235

2. Behring E, Kitasato S (1890) Über das Zustandekommen der 27. http://www.rki.de/DE/Content/Institut/Geschichte/Dokumente/G

Diphterieimmunität und der Tetanusimmunität bei Thieren. Dtsch eschichte_im_Ueberblick.pdf
Med Wochenschr 16:1113 28. Bonah C, Gradmann C (2009) Das Robert Koch Institut als Insti-
3. Klemperer G, Klemperer F (1891) Versuche über Immunisierung tution zwischen Kaiserreich und Nationalsozialismus. Interna-
und Heilung bei der Pneumokokkeninfektion. Berliner Klin tionale Beziehungen, verwaltete Wissenschaft und NS-System -
Wochenschr 28:833–835 Ein Kommentar. In Hulverschmidt A, Laukötter A (eds) Infektion
4. Fraenkel A (1886) Weitere Beiträge zur Lehre von den Mik- und Institution. Wallstein, Göttingen, pp 250–260
rokokken bei der genuinen fibrinösen Pneumonie. Z Klin Med 29. Robert Koch-Institute Archive, Berlin, Germany. Fred Neufeld
5(6):437–458 personal file, Fred Neufeld document collection (Uncatalogued)
5. Neufeld F, Händel L (1909) Über die Herstellung von Pneu- 30. Rockefeller Archive Center, Simon Flexner correspondence.

mokokkenserum und über die Aussichten einer spezifischen American Philosophical Society, Philadelphia, PA, USA
Behandlung der Pneumonie. Z Immunitätsforsch III:159–171 31. Neufeld F (1927) Origin and dissemination of tuberculosis

6. Neufeld F, Händel L (1909) Über die Entstehung der Krise bei according to recent investigations. Harvey Lect 22:27–40
der Pneumonie und über die Wirkung des Pneumokokkenserums. 32. Neufeld F (1927) Fortschritte und Rückschritte der epidemiolo-
Arb Kais Gesundheitsamt 34:166–181 gischen Forschung. Dtsch Med Wochenschr 53:687–690
7. Neufeld F, Händel L (1909) Weitere Untersuchungen über 33. Mendelsohn JA (1998) From eradication to equilibrium. how

Pneumokokken-Heilserum III Mitteilung: Über Vorkommen und epidemics became complex after world war I. In: Lawrence C,
Bedeutung atypischer Varietäten des Pneumokokkus. Arb Kais Weisz G (eds) Greater than the Parts: Holism in Biomedicine,
Gesundheitsamt 34:293–304 1920–1950, Oxford University Press, New York, pp 303–331
8. Podolsky SH (2006) Pneumonia before antibiotics. Johns Hop- 34. Kleine F (1947) Nachruf für Fred Neufeld. Med Mikrobiol

kins University Press, Baltimore Immunol 127:185–186
9. Heidelberger M, Avery OT (1923) The soluble specific substance 35. Neufeld F (1900) Ueber eine spezifische bakteriolytische

of pneumococcus. J Exp Med 38(1):73–79 Wirkung der Galle. Med Microbiol Immunol 34(1):454–464
10. Heidelberger M, Avery OT (1924) The soluble specific substance 36. Neufeld F (1902) Ueber die Agglutination der Pneumokokken
of pneumococcus: second paper. J Exp Med 40(3):301–317 und über die Theorien der Agglutination. Med Microbiol Immu-
11. Avery OT, MacLeod CM, McCarty M (1944) Studies on the nol 40(1):54–72
chemical nature of the substance inducing transformation of 37. Armstrong RR (1932) Immediate pneumococcal typing. Br Med
pneumococcal types: induction of transformation by a desoxyri- J 1(3708):187–188
bonucleic acid fraction isolated from pneumococcus type III. J 38. Logan WR, Smeall TJ (1932) A direct method of pneumococcal
Exp Med 79(2):137–158 typing. Br Med J 1(3708):188–189
12. Dochez AR (1912) The presence of protective substances in
39. Neufeld F, Etinger-Tulczynska R (1933) Schnelldiagnose der

human serum during lobar pneumonia. J Exp Med 16(5):665–679 Pneumokokkentypen aus dem Auswurf. Med Microbiol Immunol
13. Dochez AR, Avery OT (1915) Varieties of pneumococcus and 115(2):431–435
their relation to lobar pneumonia. J Exp Med 21(2):114–132 40. Neufeld F, Rimpau W (1904) Über die Antikörper des Strepto-
14. Avery OT, Chickering HAT, Cole R, Dochez AR. (1917) Acute kokken und Pneumokokken Immunserums. Dtsch Med Wochen-
Lobar Pneumonia. Prevention and Serum Treatment. Mono- schr 40:1458–1460
graphs of the Rockefeller Institute for Medical Research No. 7 41. Neufeld F, Rimpau W (1905) Weitere Mittheilungen über die
15. Wilson GS, Miles AA (eds) (1955) Topley and Wilson’s princi- Immunität gegen Streptokokken und Pneumokokken. Med
ples of bacteriology and immunity, vol 1 and 2. Edward Arnold, Microbiol Immunol 51(1):283–299
London 42. Silverstein AM (1989) A history of immunology. Academic, San
16. MacLeod CM, Hodges RG, Heidelberger M, Bernard WG (1945) Diego
Prevention of pneumococcal pneumonia by immunization with 43. Neufeld F (1903) Ueber Immunität und Agglutination bei Strep-
specific capsular polysaccharides. J Exp Med 82:445–465 tokokken. Med Microbiol Immunol 44(1):161–182
17. McCarty M (1985) The transforming principle. Norton, New York 44. Kindborg A (1905) Die Pneumokokken. Zeitschr f Hyg 51:197–232
18. Dubos R (1976) The professor, the institute, and DNA. Rockefel- 45. Römer P (1902) Experimentelle Grundlagen für klinische Ver-
ler University Press, New York suche einer Serumtherapie des Ulcus corneae serpens nach
19. Downie AW (1972) Pneumococcal transformation—a backward Untersuchung über Pneumokokkenimmunität. Graefe’s Archiv
view, fourth griffith memorial lecture. J Gen Microbiol 73:1–11 Klin Exp Ophthalmol 54:99–200
20. Olby R (1974) The path to the double helix: the discovery of 46. Dochez AR (1912) The occurrance and virulence of pneumococci
DNA. Dover, New York in the circulating blood during lobar pneumonia and the suscep-
21. Griffith F (1928) The significance of pneumococcal types. J Hyg tibility of pneumococcus strains to univalent antipneumococcus
27:113–159 serum. J Exp Med 16(5):680–692
22. Mozew C (2003) Fred Julius Neufeld (1869–1945). Sein Leben 47. Neufeld F, Ungermann E (1912) Über experimentelle Erzeugung
und Werk. Dissertation, University of Heidelberg von Pneumonien bei Versuchstieren und ihre Beeinflussung durch
23. Neufeld F (1946) Erinnerungen aus meiner 50jährigen Tätigkeit spezifische Seren. Berl Klin Wochenschr 8:717
als Bakteriologe. Ärztl Wochenschr 1(146–150):186–189 48. Neufeld F, Ungermann E (1912) Weitere Heilversuche mit Antip-
24. Neufeld F (1899) Ueber die Züchtung der Typhusbacillen aus neumokokkenserum bei experimenteller Pneumonie. Berl Klin
Roseolaflecken nebst Bemerkungen über die Technik bakte- Wochenschr 20:1010–1011
riologischer Blutuntersuchungen. Med Microbiol Immunol 49. Neufeld F, Händel L (1912) Zur Frage der Serumtherapie der
30(1):498–510 Pneumonie und der Wertbestimmung des Pneumokokkenserums.
25. Hinz-Wessels A (2008) Das Robert Koch-Institut im Nationalso- Berl Klin Wochenschr 8:680–683
zialismus. Kulturverlag Kadmos, Berlin 50. Tyler GE (1901) Antipneumococcic serum treatment of pneumo-
26. Hinz-Wessels A (2009) Das RKI unter der NS-Diktatur: Per- nia, with report of cases. J Am Med Assoc 36:1540–1545
sonelle, administrative und inhaltliche Umgestaltung zwischen 51. Goldsborough BW (1902) A contribution to the treatment of

1933 und 1945. In: Hulverschmidt A, Laukötter A (eds) Infektion pneumonia with antipneumococcic serum. J Am Med Assoc
und Institution. Wallstein, Göttingen, pp 67–88 38:1681–1683

13
2236 K. Eichmann, R. M. Krause

52. Anders JM (1904) Serum treatment of pneumonia. J Am Med 61. Potter NB, Avery OT (1910) Opsonins and vaccine therapy. In:
Assoc 43:1777–1781 Hare R (Ed) Modern Treatment. Philadelphia and New York,
53. von Hippel A (1908) Der gegenwärtige Stand der Pneumokok- 1:515
kus Serumtherapie des Ulcus serpens. Dtsch Med Wochenschr 62. Lister FS (1916) An experimental study of prophylactic inocu-
34:1805–1807 lation against pneumococcal infection in the rabbit and in man.
54. Beltz L (1912) Über die intravenöse Anwendung des Pneu-
Publ South Afr Institute Med Res 8:231–287
mokokkenserums. Dtsch Med Wochenschr 38:14–15 63. Centers for Disease Control and Prevention (2010) Prevention
55. Neufeld F (1922) Neue Forschungsergebnisse über Pneumonie. of pneumococcal disease among infants and children —use of
Dtsch Med Wochenschr 48:51–54 13-valent pneumococcal conjugate vaccine and 23-valent pneu-
56. Neufeld F (1938) Die experimentellen Grundlagen der Serumbe- mococcal polysaccharide vaccine. MMWR 59(MMWR):1–18
handlung der Pneumonie. Dtsch Med Wochenschr 64:219–221 64. Centers for Disease Control and Prevention (2012) Licensure of
57. Neufeld F, Bär F (1940) Untersuchungen über die Wirkungsweise 13-valent pneumococcal conjugate vaccine for adults aged 50
der Sulfonamide. Med Microbiol Immunol 123(1):116–125 years and older. MMWR 61:394–395
58. Neufeld F, Levinthal W (1928) Beiträge zur Variabilität der Pneu- 65. Ridda I, Musher DM (2012) Is there a potential role for protein-
mokokken. Z f Immunitätsforsch 55:324–340 conjugate pneumococcal vaccine in older adults? Australas Med J
59. Wright AE (1903) A lecture on therapeutic inoculations of bac- 5(4):231–235
terial vaccines and their practical exploitations in the treatment 66. Miyaji EN, Oliveira MLS, Carvalho E, Ho PL (2012) Sero-

of disease: delivered at the medical graduates college and poly- type-independent pneumococcal vaccines. Cell Mol Life Sci.
clinic. Br Med J 1(2210):1069–1074 doi:10.1007/s00018-012-1234-8
60. Wright AE (1910) Vaccine Therapy—its administration, value,
and limitations. Proc R Soc Med 3(Gen Rep):1–38

13