The relatively recent development and adoption of spot-based assays of immune cells has facilitated the study of human immune responses [32
]. Significant value was added by the development of a simple, antigen-independent method to stimulate resting MBC to become cells that secrete IgG [24
]. These approaches allow us to measure the circulating frequencies of both effector ASC as well as MBC [24
]. The method to reactivate MBC has been shown to cause the differentiation of CD27+ CD38+ (CD19+ CD3−) B cells into CD27+ CD38+++ ASC [24
]. Existing ASC present at the start of the culture are lost during the culture period (Crotty, personal communication), and sorting of CD3− CD19+ cells by expression of CD27 and CD38 shows that the culture conditions induce antibody secretion by only the CD27+ CD38 low cells [9
]. In our hands, we cannot explicitly rule out carryover of ASC from the start of the culture; however, ASC frequencies were not high, making carryover less likely. What are less clear are the relationships between serum IgG, and the prevalence of ASC and MBC in the blood. This study and others show HA-specific ASC and MBC (as a function of total IgG secreting cells) increased in frequency at 7 days after vaccination [9
]. In turn, the analogous MBC per million PBMC was highest at 28 days, consistent with the expansion or perisitence of a pool of circulating HA specific memory cells. However, we should note the difficulty in relating the frequency values to the actual numbers of antigen-specific cells in the blood using the MBC assay. Proliferation of selected cells during the 6-day MBC restimulation is not easily assessed, making it difficult to directly and accurately extrapolate the values back to the blood sample. Nevertheless, these observations support the concept that immunization with potential pandemic or emerging influenza HA protein in human subjects results in the development of both short and long term B cell mediated cellular immunity.
The generation of long-lived antigen-specific memory cells is a hallmark of successful vaccination [35
]. However, few studies are available that evaluate existing cellular memory responses, or those elicited by vaccination. Fewer still compare one vaccination approach to another. Instead, evaluating the success of vaccination depends on measuring serum antibody levels. In the case of influenza, assays are available to measure total influenza specific antibody (ELISA), as well as biologically active antibodies that inhibit viral agglutination of red blood cells (HAI), or neutralization of live virus (microneutralization or plaque reduction assays). The latter are considered the “gold-standard” for influenza vaccines as they have been shown to correlate well with observed rates of protection from disease. One of the reasons these assays correlate better than the ELISA based approach is that the ELISA can detect antibodies to other determinants on the protein that, while antigenic, do not necessarily confer protection from infection. Interestingly, these non-neutralizing determinants tend to be more conserved among different hemagglutinin serotypes [36
], and conserved neutralizing antigenic determinants in the HA protein have been described [40
The B cell ELISPOT assay is essentially analogous to the ELISA assay in that it will detect B cells secreting IgG whether or not those secreted antibodies will neutralize the virus or inhibit agglutination. On this basis, one could question the clinical relevance of the approach in studying vaccine related immune responses. However, it is important to consider that a general increase in the prevalence of a memory B cell population should correlate well with long-term persistence of an immune response. To be fair, there is insufficient data in the context of human diseases to know whether detecting these cellular responses can be predictive of future immune responses to modified vaccines or drifted viruses. It is also not known in humans how the presence of a memory B cell population affects subsequent immune responses to infection or vaccination with related, but not identical variants of the virus. One could imagine that the MBC could improve antigen presentation to CD4 T cells for example.
Immune responses in adults to experimental H5 influenza vaccines have been weak at best, even after two or three immunizations with up to 90µg of the same HA [26
], with proportions of subjects mounting increased serum antibody to H5 after vaccination in the range of 40–55% [26
]. Interestingly, in adult subjects receiving experimental H7 and H9 based subunit vaccines, the response rates were similarly low [43
], though recent approaches with live attenuated candidates were more successful [46
]. For comparison, in children who are seronegative, two 7.5µg doses of TIV is effective at eliciting clinically significant serum antibody titers over 80% of recipients [48
]. This suggests that inactivated subunit H5 influenza vaccines are poorly immunogenic in humans, though they may still be influenced by the immune status of the individual.
The success of seasonal influenza vaccines against H1 and H3 strains of the virus may be high because they depend on stimulating existing immunity to the viruses. Almost all individuals have been exposed to seasonal influenza virus by the age of 5 [51
]. Adults have likely had several encounters with influenza of both subtypes, and it is likely that the strong immunity elicited by infection facilitates good immune responses to the low amounts of protein (15µg of each HA) administered without adjuvant in the present trivalent inactivated subunit vaccine (TIV).
On the other hand, rHA or inactivated subunit H5 vaccines administered experimentally to human subjects have a reputation of being poorly immunogenic, based largely on standard HAI and MN titers from serum samples. The data herein supports the notion that, although neutralizing or inhibitory antibodies are weak or undetectable, vaccination can elicit long-lived cellular immunity in human subjects. In a related study of H5 vaccination, Ali-Goji et al recently showed that subjects previously vaccinated with two doses of an experimental, recombinant protein vaccine against avian H5 influenza A/Hong Kong/156/97 subsequently mounted robust responses to an experimental egg-derived H5 vaccine based on a more recent influenza A/Vietnam/1203/04 strain of the virus [26
]. Response rates determined by conventional HAI and MN assays were 86% or greater, with relatively high titers. This suggested that, although the response rates after administration of the H5/97 vaccine alone were much less than 80% [25
], the vaccine may have established a component of long-lived memory that persisted. Therefore, the possibility exists that prior vaccination may have a clinical benefit, in spite of difficulty in detecting substantial neutralizing antibody responses.
The reasons that vaccination against H5, whether in recombinant or inactivated subunit forms, does not elicit significant amounts of neutralizing antibody, while viral infection does, are not known. It may be that conformational differences exist in the purified recombinant and inactivated subunit-derived proteins versus those produced by infected cells. In the range of antibodies that are elicited, only a small subset may have neutralizing activity. It may also be the case that there are aspects of how the HA antigen is presented during infection that competitively favors the production of neutralizing antibody, versus antibody to other determinants. Nevertheless, it seems that having B cells and antibody to other determinants should confer some protection from severe disease in animals, though it would not prevent infection.
We believe that the observations lend support the idea that the phenomenon of Original Antigenic Sin may not apply to protein vaccination [9
], and is a unique feature of infection. The mechanisms that are unique to priming by infection and responsible for this effect are not known, but may explain the problem of extrapolating the concept of OAS to influenza vaccination (versus infection). Experimental HA drift variant immunization of naïve mice, for example, elicits antibodies that cross-reacted with HA of the same H5 subtype, but not across other H3 and H6 HA (data not shown), producing a similar quality of antibody responses seen in the human subjects. Unlike the mice however, the adult human subjects of the present study were not naïve to influenza, and had likely developed immunity to influenza through a history of both infection and vaccination. Unfortunately, it is not possible to resolve these issues within the context of the present study.
These studies support the observations reported for serum HAI and MN responses [26
] that suggested some advantages to pre-pandemic vaccination with vaccine candidates closely related to the anticipated pandemic strain. A certain level of durable, long-lived cellular immunity is established and reactivated by HA protein or subunit immunization, and can develop in the absence of evidence of the serological response. These cell mediated immunity (CMI) assays compliment serum assays, though the predictive value to protection from infection remains to be established. It is worth considering the expansion of these types of clinical studies to further optimize the experimental design, look at responses to other types of immunization and infection, as well as determine whether or not they will be of use in predicting vaccine success or diagnosis of infection.