Influenza causes 36,000 deaths annually in the United States alone and the influenza pandemic of 1918 caused an estimated 50 million deaths worldwide2
. Outbreaks of avian influenza infections in human populations that caused substantially higher mortality rates foresee the possibility of another deadly pandemic3
. The challenge of influenza has long been to design vaccines that induce long lasting immunity against a pathogen that rapidly alters its appearance to the immune system by mutating (antigenic drift) and exchanging (antigenic shift) its components. Antibodies play a key role in protection against influenza infection4-7
. However, the underlying B cell response leading to the rapid production of ASCs that secrete antibodies is only beginning to be understood8-12
. Critically, we do not yet know if B cell memory can provide sufficient protection early in the response to counteract variant strains of influenza or if rather the response is dominated by antibodies previously generated against divergent viruses in an OAS fashion. Finally, of profound clinical significance is the possibility that the early ASC response observed after immunization can be exploited to rapidly generate therapeutic or diagnostic mAbs to emerging influenza virus strains, or in fact to any immunizing antigen.
In order to determine the dynamics and magnitude of the human anti-influenza response we analyzed the frequency of ASCs and memory B cells in a time-course following vaccination. The ASC response was quite transient, peaking at approximately day seven and returning to barely detectable levels by day 14 after vaccination (). The frequency of influenza-specific ASCs averaged 6.4% (or ~2,500 ASCs per ml of blood) at day 7, and accounted for up to 16% of all B cells (range for ten donors: 1.1-16%, ). Also, most of these ASCs were generated during the vaccination response as they were almost entirely Ki-67 positive, indicating recent proliferation, and most expressed homogenously high levels of HLA-DR13
(). Importantly, analysis of IgG secreting ASCs isolated by cell sorting at day 7 post-immunization demonstrated that the vast majority were influenza vaccine specific (ranging from 20-85% and averaging 70%, ). The ASCs were mainly IgG positive, with minor components of IgA and IgM positive cells (data not shown), suggesting an origin from the memory B cell compartment. The memory B cell response was also quantified14
. Increasing from low levels prior to vaccination, influenza-specific memory B cells peaked a week after the ASC response at 14 to 28 days after vaccination and averaged 8.2% of the IgG+
memory B cells or ~1% of all B cells (). We conclude that influenza vaccination results in a massive burst of IgG+
ASCs that are predominantly influenza-reactive and peak at approximately day 7 post-immunization.
Analysis of the B cell response induced by influenza vaccination
The rapid accumulation of ASCs suggests that the response could be highly clonal in nature, limiting the early influenza response. Some clonal activation of ASCs occurs after tetanus vaccination12
. We therefore analyzed the immunoglobulin repertoire breadth (i.e., the variable genes and junctional diversity) of the influenza specific ASCs. Influenza vaccination caused a surprisingly pauci-clonal response, with some donors being dominated by the progeny of only a few expanded B cell clones (Supp. Fig. 1a
and Fig. 2a
). Clonal expansions accounted for 43% of the ASC variable regions from the 14 immunized donors including three donors with over 70% clonality (). In stark contrast, based on VH
regions sequenced from our laboratory in a comparable fashion15-17
, naïve and memory B cells (IgM or IgG) isolated from blood were rarely or never clonal, while for tonsillar B cells only 10% of IgM and 12% of IgG GC and memory cells were clonally related.
The ASC response after influenza vaccination is pauci-clonal and highly diversified by somatic hypermutation
Immunoglobulin variable gene somatic hypermutation allows for the generation of high affinity antibodies18,19
. Surprisingly, the influenza specific ASCs had accumulated more somatic mutations than any normal population of B cells. Considering the various donors (), the ASCs averaged 19.4 ± 3.5 VH
gene mutations, which is greater than that of germinal center or memory B cells that average 13.6 ± 4.8 mutations for IgG or 8.4 ± 3.8 mutations for IgM. A surprising 11% (41/405) of the ASC VH gene segments have more than 30 of 300 (or ~10%) of the total nucleotides altered (). A preference for CDR replacement mutations suggests that the ASCs were functionally selected (Sup. Table 1
). The observations herein suggest that the origin of the anti-influenza ASCs is predominantly memory B cells that probably accumulated new mutations on this and on previous rounds of activation.
It is not known how often the ASCs that are induced by vaccination produce high affinity antibodies against influenza. Immunoglobulin variable region genes from ASCs can be used to express specific antibodies20
. We therefore used the variable gene transcripts of isolated single ASCs to express recombinant monoclonal antibodies in the human 293 cell line (Sup. Fig. 1b
and methods). From day 7 post vaccination ASCs of five donors, 71% (61/86) of the antibodies bound with high affinity to either native antigens of the influenza vaccine strains (53/86 or 61%), or to components of the vaccine only (8/86 or 9%) (, and Sup. Fig. 2
). We suspect that the epitopes found only in the vaccine are exposed on the fixed virions or are from added preservatives. In comparison, none of the 86 mAbs generated from naïve B cells15
() and only one of 54 antibodies from random IgG memory B cells bound to the influenza vaccine strains with appreciable affinity (data not shown). The antibodies produced from the influenza specific ASCs bound to any of the three vaccine components with similar frequency ( and Sup. Fig. 2
). Analysis of viral antigen specificity by immunoprecipitation and Western blot (Sup. Fig. 3
) found that 60% of the influenza-reactive antibodies bound to HA, of which half were hemagglutination-inhibiting ( and ). Twelve percent of the antibodies bound to neuramininidase (NA) or to other minor components of the vaccine likely residual to the purification of HA and NA during vaccine production. Ten percent of the antibodies did not precipitate native antigens and bound only to epitopes on denatured viral proteins detectable by Western blot. Importantly, each of three representative HAI+ antibodies against influenza-A (anti-H3N2) and one against influenza-B from the day 7 ASCs (, bold) were found to neutralize viral infection of MDCK cells in vitro
(each neutralized virus at <1ug/ml antibody, Sup. Fig. 4
). In conclusion, after influenza vaccination early ASCs produce functional antibodies that bind with high affinity and likely provide early protection.
High affinity mAbs generated from single influenza specific ASCs
Characteristics of anti-Influenza antibodies
Although most of the ASCs arise only after vaccination (), twenty-nine percent of the antibodies generated did not detectably bind to the influenza strains or whole vaccine (). Possible causes include errors introduced by the RT-PCR steps (though PCR errors were rare, Sup. Table 2
), targeting of non-viral or denatured components of the vaccine or antigens only evident physiologically, bystander activation of non-specific memory cells8
, or displacement of non-specific plasma cells from the bone marrow13
. The latter possibility is unlikely as expression of HLA-DR13
and Ki-67 () by the ASCs suggests they were newly generated.
The long held theory of OAS suggests that new influenza variants will evade surveillance when memory B cells reactive to previous viral strains dominate the response1
. In order to consider the impact of OAS directly, we compared the relative affinity to either the current B strain virus (B/Malaysia/2506/2004) or to the two previous ones (B/Shanghai/361/2002 or B/Hong Kong/33/2001, ). In the 2006/7 season, antibodies were analyzed from five donors that had also been vaccinated in the 2005/6 season and one in 1991 so that reactive memory cells should be readily available for an OAS response. Importantly, each of the 19 anti-B strain antibodies bound to the new B strain with equal, and in most cases with greater affinity than the previous vaccine strains ( and Sup. Fig. 2
). This adaptation occurred despite the only 10% or less difference of the HA sequence of the 2006/7 B strain from those used in previous vaccines. Although previous exposure to B/Malaysia/2506/2004 cannot be entirely ruled out, there was no history of exposure and pre-vaccination serum titers of antibody against B/Malaysia/2506/2004 were not above background levels (data not shown). Thus we conclude that even for the earliest detectable influenza-specific B cells after vaccination, the ASCs, OAS does not limit reactivity to newly introduced influenza strains.
Specificity for the newly introduced influenza B strain in the vaccine suggests a minimal impact of OAS
In conclusion, we show that after influenza vaccination we can isolate an almost entirely antigen specific population of ASCs that comprise about 5% of all blood-borne B cells. Further, the findings herein help to resolve a major obstacle longstanding in the field of medicine21
: the rapid production of fully human monoclonal antibodies. Antibody or serum therapy has been demonstrated to effectively treat a plethora of diseases, but is not widely used because sometimes fatal anaphylactic responses and serum sickness are common. These obstacles can only be overcome by using fully human mAbs. The findings herein demonstrate that we can now generate human monoclonal antibodies from the antigen-specific ASCs directly, and within only weeks of vaccination (Supp. Fig. 1C
). With a modern resurgence of interest in monoclonal antibody therapy we anticipate that antibodies produced from post-vaccination ASCs will generate substantial advances for the treatment of infectious diseases.
Conventional wisdom holds that the level of pre-formed antibody is the main correlate of protection against influenza virus. However, our results showing the rapidity of the antibody response after vaccination and the high affinity of the antibodies produced strongly suggests that the recall response could also play a role in protective immunity. This antibody would, of course, not prevent initial infection but could play a crucial role in preventing the spread of virus and bringing about faster resolution of the infection. This notion is supported by our finding that OAS was not a significant aspect of the memory response as the antibodies produced were highly specific to the immunizing antigen.