Drs. Nikolic, Tan, and their colleagues present an interesting paper demonstrating the efficacy of transcutaneous (TC) immunization with Aβ1-42 peptide and cholera toxin (CT) adjuvant. Wild-type and PSAPP (APPsw/PSEN1dE9) mice were immunized weekly for 4 weeks and then biweekly for an additional 12 weeks. Anti-Aβ titers were first detected at 4 weeks and rose thereafter. Anti-Aβ antibodies were of mostly IgG1 (Th2) isotypes, but lower levels of IgG2a (Th1) and IgG2b (Th2) antibodies were also generated. Splenocytes from Aβ/CT immunized mice secreted dramatically elevated levels of IL-4 along with less dramatic elevations in IFN-γ and IL-2 compared to CT-treated control mice. PSAPP mice were immunized from 4 to 8 months of age and showed significant reductions in cerebral plaque burden, soluble Aβ40 and Aβ42, and insoluble Aβ40 and Aβ42. Plasma Aβ was increased. T cell infiltration and microhemorrhage were not observed in any of the animals. The authors conclude that transcutaneous immunization with Aβ is effective in lowering cerebral Aβ (perhaps by a peripheral sink mechanism)...  Read more

Drs. Nikolic, Tan, and their colleagues present an interesting paper demonstrating the efficacy of transcutaneous (TC) immunization with Aβ1-42 peptide and cholera toxin (CT) adjuvant. Wild-type and PSAPP (APPsw/PSEN1dE9) mice were immunized weekly for 4 weeks and then biweekly for an additional 12 weeks. Anti-Aβ titers were first detected at 4 weeks and rose thereafter. Anti-Aβ antibodies were of mostly IgG1 (Th2) isotypes, but lower levels of IgG2a (Th1) and IgG2b (Th2) antibodies were also generated. Splenocytes from Aβ/CT immunized mice secreted dramatically elevated levels of IL-4 along with less dramatic elevations in IFN-γ and IL-2 compared to CT-treated control mice. PSAPP mice were immunized from 4 to 8 months of age and showed significant reductions in cerebral plaque burden, soluble Aβ40 and Aβ42, and insoluble Aβ40 and Aβ42. Plasma Aβ was increased. T cell infiltration and microhemorrhage were not observed in any of the animals. The authors conclude that transcutaneous immunization with Aβ is effective in lowering cerebral Aβ (perhaps by a peripheral sink mechanism) and may be safer than the original Aβ vaccines tested in mice and humans because Langerhans cell precursors, a major antigen presenting cell in skin, drive a predominantly Th2 immune response.

The data presented here bode well for a transcutaneous delivery of an Aβ vaccine for Alzheimer disease. They confirm, to some degree, data we reported last year in which transcutaneous immunization with a short Aβ immunogen (dendrimic Aβ1-15: 16 copies of Aβ1-15 on a lysine tree) and adjuvant LT(R192G) resulted in Aβ antibody generation in wild-type mice (Seabrook et al., 2006). Anti-Aβ levels and isotypes were similar between the two studies.

Interestingly, in our study, TC immunization with full-length Aβ peptide did not lead to anti-Aβ production. This inconsistency with Nikolic's paper may be due to differences in adjuvant (CT vs. LT), dose (200 µg Aβ1-42 vs. 100 µg Aβ1-40/42), schedule (weekly for 4 weeks then biweekly for 12 weeks vs. biweekly for 16 weeks), and/or slight differences in the transcutaneous application of the vaccine.

We did not report results for TC immunization with dAβ1-15 in AD mouse models; however, we believe that by avoiding an Aβ-specific T cell epitope in our immunogen, the vaccine may avoid an Aβ-specific T cell autoimmune response, such as that suggested to be responsible for the adverse events in the AN-1792 trial using full-length Aβ1-42 peptide. In our study, splenocytes from wild-type mice TC immunized with dAβ1-15 did not recognize full-length Aβ, as they did not proliferate upon restimulation with Aβ peptide. In the Nikolic et al. paper, splenocytes from wild-type and PSAPP mice TC immunized with Aβ/CT recognized full-length Aβ, resulting in increased levels of both Th2 and Th1 cytokines. Whether or not such a response in humans would predispose to an Aβ autoimmune response remains unclear.

In the current paper, the authors found no T cell recruitment to brain; however, most Aβ vaccines studies in mice have shown the same result. It has been difficult to replicate in mice the adverse events observed in the human trial. Only a couple of papers have reported T cell recruitment to the brain upon Aβ immunization, and only when pertussis toxin was co-administered with the vaccine and again 2 days later (Furlan et al., 2003; Monsonego et al., 2006). In particular, Monsonego used an Aβ T cell epitope, Aβ10-24, for his immunogen to immunize APP double transgenic mice that overexpressed IFN-γ. Thus, in the present paper, it is not completely surprising that T cells were not recruited to brain. The need remains to test these vaccines (including ours) in an animal model that mimics the effects seen in the human subjects in the AN-1792 trial.

Lastly, microhemorrhage has been associated with passive transfer of Aβ antibodies in older APP or PSAPP transgenic mice bearing cerebrovascular amyloid angiopathy (CAA) at the time of immunization; however, it is less clear that the same effect would occur if passive immunization were to be delivered to young animals prior to plaque deposition or CAA. The current paper, in which mice were first immunized prior to plaque deposition, suggests that the lack of microhemorrhage indicates safety; however, older mice with CAA should be examined. To date, we have not observed microhemorrhage with any of our active Aβ vaccines when given in a "prevention" trial in which the immunization began prior to plaque deposition.

In summary, the authors are to be congratulated on the successful generation of anti-Aβ antibodies and lowering of cerebral Aβ in their study of transcutaneous immunization in PSAPP transgenic mice. Further studies are now needed to assess the safety issues raised. We agree with the authors that an effective and safe transcutaneous Aβ vaccine would be convenient, less costly, and less painful than an injectable vaccine that requires a doctor's office visit.

View all comments by Cynthia Lemere

The skin is a well-established effective route for vaccination. The authors evaluate the efficacy of transcutaneous immunization of PSAPP Tg mice in reducing cerebral amyloidosis using aggregated Aβ1-42 plus cholera toxin. Reduction in cerebral amyloidosis was not associated with deleterious side effects including brain T cell infiltration or cerebral microhemorrhage.

Previous studies (Schenk et al., 1999) showed that this antigen works in mice, but it then proved to be dangerous to humans. Intraperitoneal immunization with Aβ1-42 generates antibodies, particularly against the N-terminal of the peptide, the immuno-dominant region of the Aβ peptide; therefore, it is not necessary to use whole peptide, which may induce an inflammatory T cell response. On the basis of endogenous reactivity to Aβ in patients with AD, the use of a full-length Aβ peptide might be expected to lead to T cell-mediated CNS inflammatory effects. Increased T cell reactivity to Aβ was not observed preclinically in APP transgenic mice, perhaps due to their...  Read more

The skin is a well-established effective route for vaccination. The authors evaluate the efficacy of transcutaneous immunization of PSAPP Tg mice in reducing cerebral amyloidosis using aggregated Aβ1-42 plus cholera toxin. Reduction in cerebral amyloidosis was not associated with deleterious side effects including brain T cell infiltration or cerebral microhemorrhage.

Previous studies (Schenk et al., 1999) showed that this antigen works in mice, but it then proved to be dangerous to humans. Intraperitoneal immunization with Aβ1-42 generates antibodies, particularly against the N-terminal of the peptide, the immuno-dominant region of the Aβ peptide; therefore, it is not necessary to use whole peptide, which may induce an inflammatory T cell response. On the basis of endogenous reactivity to Aβ in patients with AD, the use of a full-length Aβ peptide might be expected to lead to T cell-mediated CNS inflammatory effects. Increased T cell reactivity to Aβ was not observed preclinically in APP transgenic mice, perhaps due to their high levels of peripheral Aβ and the consequent induction of T cell tolerance (Monsonego et al., 2001).

Improved immunotherapeutic strategies may be used to obtain beneficial effects without untoward side effects. It is possible to induce Aβ antibodies with no T cell response using small peptides containing dominant B cell epitopes that bind to Aβ plaques but no T cell epitopes; it will be equally possible to find the most convenient way of delivery.

View all comments by Beka Solomon

I would like to comment on the following statement from this News Article:

"It is important to note that the original vaccine did not produce T cell infiltration in mice, either, yet still caused problems in some people."

I wonder how good the evidence is supporting the idea that the original vaccine (or the new skin vaccine) does not produce T cell infiltration in mice. How many mice would have to be examined (and how carefully?) to rule out an effect that only occurred in ~6 percent of subjects in the AN1792 trial?

View all comments by DAVID HAWVER

We appreciate the comments of Drs. Lemere and Solomon. I'd like to comment on a few of the issues they raised.

Regarding cerebral microhemorrhage, it is correct that this adverse event has been observed in past "active" Aβ vaccines that have been administered to old transgenic AD mice bearing established amyloid plaques. So, I agree with Dr. Lemere that lack of detection of these microhemorrhages is not altogether unexpected in our transcutaneous Aβ vaccine, which was administered prophylactically starting in younger AD mice. I also agree that it has been difficult to understand why Aβ immunization does not produce the aseptic meningoencephalitis in AD mice that was observed in about 6 percent of patients who received Elan's AN-1792 vaccine (unless pertussis toxin is used as the adjuvant—often employed to promote brain T cell infiltration in mouse models of multiple sclerosis). Ultimately, this may come down to differences in immune systems of mice versus humans. It may be necessary to use mice with "humanized" immune systems (currently under development by Dr. Richard...  Read more

We appreciate the comments of Drs. Lemere and Solomon. I'd like to comment on a few of the issues they raised.

Regarding cerebral microhemorrhage, it is correct that this adverse event has been observed in past "active" Aβ vaccines that have been administered to old transgenic AD mice bearing established amyloid plaques. So, I agree with Dr. Lemere that lack of detection of these microhemorrhages is not altogether unexpected in our transcutaneous Aβ vaccine, which was administered prophylactically starting in younger AD mice. I also agree that it has been difficult to understand why Aβ immunization does not produce the aseptic meningoencephalitis in AD mice that was observed in about 6 percent of patients who received Elan's AN-1792 vaccine (unless pertussis toxin is used as the adjuvant—often employed to promote brain T cell infiltration in mouse models of multiple sclerosis). Ultimately, this may come down to differences in immune systems of mice versus humans. It may be necessary to use mice with "humanized" immune systems (currently under development by Dr. Richard Flavell and colleagues) to better model this aspect of the Aβ vaccine and allow appropriate steps to be taken to avoid this harmful adverse event.

Regarding the use of N-terminal Aβ "B cell epitopes" in lieu of Aβ middle-region "T cell epitopes," it would be great if this approach ultimately works in humans (as has been shown in AD mice by Dr. Lemere), should it make it to clinical trials. However, I have my doubts. It seems that production of Aβ antibodies is the key step to producing therapeutic benefit, whether it is the ~0.1 percent of circulating Aβ antibodies that we know from the work of Schenk and colleagues to cross the blood-brain barrier in mice, or the circulating antibodies from the work of Holtzman and colleagues that may act as a "peripheral sink" to draw Aβ out of the brain and into the blood. If we use a non-T cell reactive portion of the Aβ peptide as an immunogen, B cells will not receive the T cell help necessary to elicit a fulminant Aβ antibody response—and by "fulminant" I mean IgM to IgG class-switching and copious production of long-lasting Aβ IgG antibodies.

An approach that in my opinion is more likely to succeed would be promoting anti-inflammatory, cognate Th2 cell Aβ-specific immunity. Th2 cells are more efficient than Th1 cells at providing help to B cells, and are defined on the basis of anti-inflammatory cytokines such as IL-4 and IL-10 (as opposed to pro-inflammatory Th1 cells, which may be the T cell subset that mediated the aseptic meningoencephalitis in the Elan/Wyeth trial). So, Th2 cells (unlike Th1 cells) should not provoke a pro-inflammatory, auto-aggressive immune response. We’ve seen from our past studies, as well as in studies from Dr. Lemere’s group and others, that there are different ways to promote Th2 immune responses in the context of Aβ; for example, depending on antigen route of delivery, adjuvant used, and dosing schedule. In our recent transdermal Aβ vaccine approach, we have targeted skin-resident Langerhans cells, a unique population of innate immune cells that we believe are important in promoting Th2 immune responses to Aβ. Not only is the skin a minimally invasive, convenient route of delivery for the Aβ vaccine, but it is also an organ that offers unique immunomodulatory potential.

References:
Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D, Seubert P. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 1999 Jul 8 ; 400(6740):173-7. Abstract

DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzma n DM. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A 2001 Jul 17;98(15):8850-5. Abstract

Weiner HL, Lemere CA, Maron R, Spooner ET, Grenfell TJ, Mori C, Issazadeh S, Hancock WW, Selkoe DJ. Nasal administration of amyloid-beta peptide decreases cerebral amyloid burden in a mouse model of Alzheimer's disease. Ann Neurol. 2000 Oct ; 48(4):567-79. Abstract

Maier M, Seabrook TJ, Lazo ND, Jiang L, Das P, Janus C, Lemere CA. Short amyloid-beta (Abeta) immunogens reduce cerebral Abeta load and learning deficits in an Alzheimer's disease mouse model in the absence of an Abeta-specific cellular immune response. J Neurosci. 2006 May 3;26(18):4717-28. Abstract

Town T, Tan J, Flavell RA, Mullan M. T cells in Alzheimer's disease. Neuromolecular Med. 2005 Jan 1;7(3):255-64. Abstract

Town T, Vendrame M, Patel A, Poetter D, Delledonne A, Mori T, Smeed R, Crawford F, Klein T, Tan J, Mullan M. Reduced Th1 and enhanced Th2 immunity after immunization with Alzheimer's beta-amyloid(1-42). J Neuroimmunol. 2002 Nov ;132(1-2):49-59. Abstract

Nikolic WV, Bai Y, Obregon D, Hou H, Mori T, Zeng J, Ehrhart J, Shytle RD, GiuntaB, Morgan D, Town T, Tan J. Transcutaneous b-amyloid peptide immunization of transgenic Alzheimer’s mice results in reduced cerebral B-amyloid deposits in the absence of T cell infiltration and microhemorrhage. PNAS Early Edition, week of Jan. 22.

View all comments by Terrence Town

In this interesting paper, Nikolic and colleagues examined the efficacy of transcutaneous immunization (TCI) with fAβ42 and cholera toxin (CT) in induction of immune responses to Aβ and reducing cerebral amyloidosis in PSAPP mice without development of significant amyloid deposits at the start of immunization (protective vaccination).

It is well known that the first immunotherapy clinical trial (AN-1792 vaccine) in AD patients was halted when ~6 percent of the participants developed aseptic meningoencephalitis. Case reports from AN-1792 trials suggest that the aseptic meningoencephalitis detected in 22 percent of the vaccine-responsive subgroup (59 individuals with antibody titers ^³1:2200) might have been caused by a T cell-mediated autoimmune response (Nicoll et al., 2003; Ferrer et al., 2004; Nicoll et al., 2006), although Aβ-specific CD4+ or CD8+ T cells have never been directly demonstrated in the brains. Importantly,...  Read more

In this interesting paper, Nikolic and colleagues examined the efficacy of transcutaneous immunization (TCI) with fAβ42 and cholera toxin (CT) in induction of immune responses to Aβ and reducing cerebral amyloidosis in PSAPP mice without development of significant amyloid deposits at the start of immunization (protective vaccination).

It is well known that the first immunotherapy clinical trial (AN-1792 vaccine) in AD patients was halted when ~6 percent of the participants developed aseptic meningoencephalitis. Case reports from AN-1792 trials suggest that the aseptic meningoencephalitis detected in 22 percent of the vaccine-responsive subgroup (59 individuals with antibody titers ^³1:2200) might have been caused by a T cell-mediated autoimmune response (Nicoll et al., 2003; Ferrer et al., 2004; Nicoll et al., 2006), although Aβ-specific CD4+ or CD8+ T cells have never been directly demonstrated in the brains. Importantly, AN-1792 vaccine utilized fibrillar Aβ42 (fAβ42) containing the B and T cell “self epitopes” of this peptide as an immunogen, and a Th1 adjuvant, QS21, which was also implicated as a cause of the reported adverse effects.

Our previous studies with mice also suggested that both adjuvant and T cell epitopes of Aβ42 might be critical to safe AD vaccine design (Cribbs et al., 2003). Accordingly, we suggested to circumvent the side effects of the AN-1792 vaccine and reduce the potential for T cell-mediated autoimmune toxicity in AD patients, using a vaccine composed of B cell antigenic determinants of Aβ42 fused with a “non-self” T helper cell epitope (Agadjanyan et al., 2005; Petrushina et al., 2007 submitted).

While we and others (Seabrook et al., 2006; Seabrook et al., 2006) were trying to avoid autoreactive T cell responses, Nikolic et al. have decided to keep both B cell and T cell self-epitopes using whole fAβ42 peptide, but to use the CT adjuvant instead of QS21 in order to generate an effective and potentially safe AD vaccine. CT, produced by various strains of Vibrio cholerae, is an exceptionally potent and safe immunoadjuvant not only for rodents, but also for humans, despite the fact that it induces strong anti-toxin immunity (Glenn et al., 1998; 1998; 1999). It was shown that TCI with tetanus toxoid and CT induced systemic Th2 (anti-inflammatory)-type (Hammond et al., 2001) responses, whereas CT mixed with OVA induced both Th1 (inflammatory) and Th2 responses (Anjuere et al., 2003).

Nikolic et al. analyzed the humoral immune responses at day 0 and at weeks 4, 8, 12, and 16 after TCI, and demonstrated that the first four injections were not inducing much antibody responses to self Aβ in PSAPP mice. It remains to be investigated why the first four, or maybe even five immunizations were not potent (many reports with other antigens + CT indicate that 2-3 TCI are very potent, and this is important for use of a vaccine in humans). After the sixth injection with fAβ42 and CT, mice generated very high titers of anti-Aβ42 antibodies (average titers jumped to ~120 mkg/ml ± SD of ELISA) that continued to increase after eight (~180 mkg/ml) and 10 (>200 mkg/ml) immunizations. Although the antibody response of individual mice is not reported by the authors, it is clear that average titers of >200 mkg/ml are extremely high for APP/Tg mice.

Along with these high titers of antibodies, one should expect strong anti-Aβ42 T helper cell responses in immune animals. Although the authors did not investigate proliferation of CD4-positive T helper cells in PSAPP or wild-type mice, they did analyze production of IL2 as well as IL4 (anti-inflammatory) and IFNg (inflammatory) cytokines in cultures of splenocytes obtained from C57Bl6 (Fig 1 in manuscript) and PSAPP (data were not shown in the paper) mice immunized with fAβ42 plus CT or CT alone. The authors reported folds of increase, but not concentrations of these cytokines in splenocyte cultures. The authors showed that immunizations with fAβ42 and CT, but not CT alone, induced elevation of the levels of all three cytokines after in vitro re-stimulation of cultures with fAβ42. Of note, the very strong non-specific polyclonal stimulators Con A and anti-CD3 antibodies somehow induced only a twofold increase in production of all three cytokines in experimental and control splenocyte cultures.

Taken together, these results suggest that the CT adjuvant stimulates systemic anti-Aβ Th1 and Th2 types of immune responses, and such activated T cells may recognize Aβ42 in brains of vaccinated animals and induce encephalomyelitis. To analyze this possibility, the authors tried to detect CD3 + T cells in brains of immune and control mice. They did not see lymphocyte infiltration in brains of TCI mice, but this is not surprising, because all previous studies (except one unconfirmed report with Aβ42 and pertussis toxin, i.e., Furlan, et al., 2003) also did not identify T cells in the brains of different APP/Tg mice, or wild-type animals immunized with Aβ antigen. Hopefully other AD animal models (dogs? monkeys?) could be used to show the safety of any candidate Aβ vaccine; otherwise, we will need again to go to clinical trials to demonstrate safety and efficacy of active immunization strategy in AD patients.

Having shown high titers of anti-Aβ antibodies in PSAPP mice that at the start of immunization were ~4 months old, the authors evaluated amyloid pathology after 16 weeks and demonstrated that these antibodies reduced cerebral amyloidosis in 8-month-old mice. Importantly, anti-Aβ antibodies reduced the levels of not only insoluble, but also soluble, likely more toxic forms of cerebral Aβ detected by capture ELISA. This finding is of interest because it suggests that immunotherapy for AD might not only remove amyloid deposits, but also take out most toxic, oligomeric Aβ species from the brains of AD patients. Previously, other researchers have reported that in AD patients vaccinated with AN-1792 “parenchymal amyloid was focally disaggregated” and “total soluble amyloid levels were sharply elevated in vaccinated patient gray and white matter compared with AD cases.”

Interestingly, our therapeutic vaccine induced on average >50 mkg/ml anti-Aβ antibodies in APP/Tg2576 mice (~9 months old at start of immunization), and these antibodies significantly reduced insoluble, but not soluble Aβ, including 6-, 9-, and 12-mer oligomeric species of Aβ, detected in aged ~19-month-old animals (Petrushina et al., 2007, submitted). Collectively, these results and data reported by Nikolic et al. suggest that either higher concentration of anti-Aβ antibodies are needed for significant reduction of oligomeric amyloid in brains of immunized mice, or immunization should be initiated earlier, in mice without pre-existing AD-like pathology (preventive vaccination versus therapeutic vaccination). Interestingly, data from the AN-1792 report (Patton et al., 2006) also suggest that “anti-amyloid immunization may be most effective not as therapeutic or mitigating measures, but as a prophylactic measure when Aβ deposition is still minimal.”

View all comments by Michael G. Agadjanyan

The paper by Nikolic and colleagues reports a transcutaneous method to vaccinate mice with Aβ and induce anti-Aβ antibodies. The authors found that there was an increase in anti-Aβ antibodies and a decrease in cerebral Aβ. Overall, this is an interesting study and is in agreement with our forthcoming paper in Neurobiology of Aging. In that study we also immunized mice using the transcutaneous route but used instead a short Aβ fragment containing the B cell epitope.

However, the cytokine data shown in the current manuscript also shows an increase in Th1 type cells as seen by the 4 fold increase in IFN-γ and 5 fold increase in IL-2 compared to PBS stimulation. In addition, it is often the case that IL-2 levels are higher at earlier time points of stimulation such as 24 and 48 hours, thus the peak levels of this cytokine may have been missed. I share the concern expressed in other commentaries of the very low levels of cytokines induced by ConA. Splenocyte proliferation data would also be useful to demonstrate the specificity and magnitude of the T cell response. Together,...  Read more

The paper by Nikolic and colleagues reports a transcutaneous method to vaccinate mice with Aβ and induce anti-Aβ antibodies. The authors found that there was an increase in anti-Aβ antibodies and a decrease in cerebral Aβ. Overall, this is an interesting study and is in agreement with our forthcoming paper in Neurobiology of Aging. In that study we also immunized mice using the transcutaneous route but used instead a short Aβ fragment containing the B cell epitope.

However, the cytokine data shown in the current manuscript also shows an increase in Th1 type cells as seen by the 4 fold increase in IFN-γ and 5 fold increase in IL-2 compared to PBS stimulation. In addition, it is often the case that IL-2 levels are higher at earlier time points of stimulation such as 24 and 48 hours, thus the peak levels of this cytokine may have been missed. I share the concern expressed in other commentaries of the very low levels of cytokines induced by ConA. Splenocyte proliferation data would also be useful to demonstrate the specificity and magnitude of the T cell response. Together, these data demonstrate a shift towards a Th2 type phenotype but this is not exclusive.

The quantification of the anti-Aβ antibody in the brain of approximately 0.05 percent following immunization could be explained by blood contamination following perfusion. It would be good to report the total amount of IgG detected in both Aβ/CT and CT immunized WT and PSAPP mice to allow a comparison to be made.

Based on these data and our previous work, I believe that transdermal immunization may be an effective method of immunization. However, I do not believe that it can avoid the generation of Th1 cells in humans. Humans, unlike mouse strains, express a wide variety of HLA haplotypes, and some of them are likely to generate a Th1 response to full-length Aβ. This suggests that the use of a B cell epitope-based vaccine in conjunction with a Th2 biasing adjuvant and administration route will all be required to avoid the deleterious immune response seen in the previous AN-1792 trial.

View all comments by Tim Seabrook

We would like to respond to some of the issues raised by Drs. Hawver, Agadjanyan, and Seabrook.

Regarding Dr. Hawver's point of evaluating T cells in the brains of mice that received the transcutaneous Aβ vaccine in our study, it is of course difficult to conclude that there are no T cells in the brains of these mice. We examined multiple brain sections from these immunized mice, and in parallel sections from positive control brains where mice had been induced with experimental autoimmune encephalomyelitis (day 20 after induction), a mouse model of multiple sclerosis. We easily detected T cells by CD3, CD4, and CD8 immunostaining in the latter, but not in the former. Of course, absence of proof does not constitute proof of absence, but we feel confident that there is not appreciable/significant infiltration of T cells in the brains of mice immunized transcutaneously with Aβ.

In response to Dr. Agadjanyan’s comment regarding Aβ antibody responses in C57BL/6 mice after four immunizations with Aβ plus CT, we agree that humoral responses of these mice were not strong until...  Read more

We would like to respond to some of the issues raised by Drs. Hawver, Agadjanyan, and Seabrook.

Regarding Dr. Hawver's point of evaluating T cells in the brains of mice that received the transcutaneous Aβ vaccine in our study, it is of course difficult to conclude that there are no T cells in the brains of these mice. We examined multiple brain sections from these immunized mice, and in parallel sections from positive control brains where mice had been induced with experimental autoimmune encephalomyelitis (day 20 after induction), a mouse model of multiple sclerosis. We easily detected T cells by CD3, CD4, and CD8 immunostaining in the latter, but not in the former. Of course, absence of proof does not constitute proof of absence, but we feel confident that there is not appreciable/significant infiltration of T cells in the brains of mice immunized transcutaneously with Aβ.

In response to Dr. Agadjanyan’s comment regarding Aβ antibody responses in C57BL/6 mice after four immunizations with Aβ plus CT, we agree that humoral responses of these mice were not strong until after the first four immunizations. Actually, we detected Aβ antibodies at the time of the fourth immunization, which markedly increased thereafter. As to why it took four+ immunizations to achieve Aβ antibody responses versus other reports where two to three vaccinations produced humoral responses as Dr. Agadjanyan mentions, this could be due to differences in Aβ versus other immunogens, variations in how the transcutaneous vaccine was applied, or perhaps mouse strain differences.

Drs. Agadjanyan and Seabrook raise the issue of the responses that we obtained with the non-specific stimulators ConA and CD3 antibody. Specifically, they comment that the responses that we obtained in Fig. 1D on IFN-γ, IL-2, and IL-4 cytokines were not very strong. We represented these data as fold increases over splenocytes cultured from PBS-immunized control mice. We chose to represent the data this way so that one could easily see putative enhanced cytokine release from Aβ plus CT-immunized mice and mice immunized with CT alone. We observed approximately two- to threefold more cytokines from the latter groups over the former. One would not expect that non-specific stimulation of splenocytes from the latter groups would produce a marked increase in cytokine over splenocytes from PBS-immunized mice. After all, these are non-specific mitogens; why would we expect to get a marked increase in cytokines from splenocytes cultured from Aβ plus CT or CT-immunized mice, when we are not specifically stimulating Aβ and/or CT-specific T cells? On the other hand, Aβ recall challenge does produce clear increases in cytokines in splenocytes cultured from Aβ plus CT-immunized mice.

Regarding Dr. Seabrook’s comment of measuring splenocyte proliferation, we did not conduct this assay. However, given the increase in IL-2 production after Aβ recall stimulation in splenocytes from Aβ/CT t.c. immunized mice, we may expect increased proliferation responses (as IL-2 is thought to be sufficient for T cell expansion). We appreciate Dr. Seabrook’s comment that we also observed Th1 cytokines (IFN-γ and IL-2), albeit at lower levels than Th2-indicative IL-4 production, in splenocytes from Aβ/CT t.c. immunized mice. Our interpretation of this was that given 1) IL-4 production at higher levels in vitro and 2) predominantly IgG1 Aβ antibodies in vivo, we were observing a mostly Th2-type response. The observation of appreciable IL-2 and IFN-γ from these splenocytes may represent an in-vitro phenomenon, as we did not observe evidence of a Th1-mediated humoral response in vivo.

We appreciate Dr. Agadjanyan’s comment regarding reduction in both soluble and insoluble Aβ species after Aβ/CT t.c. immunization—we were also struck by this finding and are beginning to think about a possible mechanism for this result.

Regarding Dr. Seabrook’s comment regarding blood contamination that could account for the Aβ antibodies in the brain, it is possible. However, we examined apolipoprotein B (present in blood but not normally in brain) levels in brain and could not detect this (see supplemental Fig. 7). Also, we performed Perl’s Prussian blue stain for ferric ion-hemosiderin (to detect blood contamination in brain, see Fig. 4), and we did not observe signal either in or around cerebral vessels. So we do not have evidence that poor perfusion contributed to our detection of Aβ antibodies in the brains of Aβ/CT t.c. immunized mice. Dale Schenk originally detected about 0.1 percent of Aβ antibodies in the brain (Schenk et al., 1999), and we show about half of that using our t.c. approach.

Dr. Seabrook also comments that he does not believe that t.c. immunization with full-length Aβ1-42 can avoid Th1 responses in humans because humans have more HLA haplotypes than do mice. While we agree with the latter, why would more HLA haplotypes in humans result in a Th1 response? A number of adjuvants have been used in humans, and some of them (e.g., alum, CT) consistently produce a Th2 response, irrespective of the peptide used for immunization or the HLA haplotype of the individual. Our belief is that the “danger signal” provided by the adjuvant and the immune cells targeted (i.e., route of administration) may be more important than the peptide itself in promoting Th1 versus Th2 responses. Of course, we won’t ultimately know until data are available—if ever—in humans using these different approaches. That being the case, the more approaches that are explored, the better the chance that one or more of them will ultimately work.

References:
Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D, Seubert P. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 1999 Jul 8;400(6740):173-7. Abstract

View all comments by Jun Tan
View all comments by Terrence Town

I’d like to offer for the field’s collective consideration my view that this manuscript is flawed in several regards.

First, some background on DNA-based vaccination. A unique property of DNA-based vaccination is its ability to induce prolonged, endogenous antigen synthesis and processing within the subject’s own cells. DNA immunization has been shown to generate humoral and cellular immune responses against multiple viral, bacterial, and tumor antigens as well as against amyloid-β (Aβ) self-antigen (1) as was first shown by our group. This approach allows also the rational inactivation or removal of sequences encoding potentially toxic domains as well as the inclusion of molecular adjuvants, such as chemokines, cytokines, or co-stimulatory molecules that can direct T helper cell responses toward the desired pathway.

With regard to the requirements for an Alzheimer disease (AD) vaccine, DNA immunization exhibits several important advantages when compared to peptide-based AD vaccines or passive immunization with monoclonal antibodies. For example, DNA vaccines do not...  Read more

I’d like to offer for the field’s collective consideration my view that this manuscript is flawed in several regards.

First, some background on DNA-based vaccination. A unique property of DNA-based vaccination is its ability to induce prolonged, endogenous antigen synthesis and processing within the subject’s own cells. DNA immunization has been shown to generate humoral and cellular immune responses against multiple viral, bacterial, and tumor antigens as well as against amyloid-β (Aβ) self-antigen (1) as was first shown by our group. This approach allows also the rational inactivation or removal of sequences encoding potentially toxic domains as well as the inclusion of molecular adjuvants, such as chemokines, cytokines, or co-stimulatory molecules that can direct T helper cell responses toward the desired pathway.

With regard to the requirements for an Alzheimer disease (AD) vaccine, DNA immunization exhibits several important advantages when compared to peptide-based AD vaccines or passive immunization with monoclonal antibodies. For example, DNA vaccines do not require the use of a conventional adjuvant, scale-up of manufacturing for clinical trials is readily achieved, and cGMP-grade DNA vaccines are significantly less expensive than peptide-based cGMP vaccines.

However, despite considerable promise demonstrated in animal models, DNA vaccines have generally exhibited low and inconsistent immune responses in clinical studies. There is consensus in the field that a key factor in this limitation is the low delivery efficiency of current DNA administration technologies. Two methods that can be used to increase the efficiency of DNA delivery into cells are the use of a gene gun (the only device for human use belongs to PowderMed/Pfizer) and electroporation (devices for human use belong to two companies, Ichor Medical Systems and Inovio Biomedical Corporation). Our mouse studies demonstrate that both gene gun- and electroporation-mediated delivery of DNA-based AD vaccines promote very strong antibody responses to Aβ in both wild-type and APP/Tg mouse models (1-5).

In this volume of JAMA, Lambracht-Washington et al. presented data demonstrating that multiple immunizations (the exact number is unclear from the manuscript but looks like up to 14 times) of B6SJLF1 wild-type mice with DNA encoding three copies of Aβ42 generated ~15 μg/ml antibodies. Basically, these authors repeated a pioneering study of Ghochikyan et al. (1), except they used a complicated and, in my opinion, debatable (see below) plasmid system instead of the single and effective plasmid used in our previous work published six years ago. Publication of this simple and arguably non-innovative paper in a prestigious medical journal with a 23.5 impact factor raises several questions in my mind, and I would try to address some here.

First, AD vaccine researchers want to understand if it is safe to generate B and T cell responses to a DNA vaccine encoding full-length Aβ42 peptide. It is well known that an AN1792 immunotherapy vaccine trial was halted prematurely because a subset of vaccinated, but not control, individuals developed meningoencephalitis. Postmortem examinations revealed that the neuroinflammation consisted primarily of CD4+ and/or CD8+T cells, implying that auto-reactive T cell responses to self-epitopes within the Aβ42 peptide were responsible for this serious adverse event. If that is the case, it is likely that any DNA vaccine encoding full-length Aβ42 may also generate auto-reactive T cells and induce inflammation in humans. In this study the authors claim that their DNA vaccine did not activate T cells specific to amyloid, but that raises the question of how the vaccinated mice managed to produce not high, but still substantial, humoral responses. Aβ42 is a T-dependent antigen (in our lab immunizations of nude mice of H2b and H2d haplotypes with fibrillar Aβ42 peptide formulated in CFA/IFA did not induce any humoral responses), and that is why without CD4+T helper cell responses, mice couldn’t produce antibodies. Thus, in order to move toward a clinical trial, the authors of this paper should clearly demonstrate the differences between the peptide antigen used in AN1792 and their DNA vaccine encoding three copies (probably one copy is not immunogenic) of full-length Aβ42. Another problem is that not every kind of animal model is suitable for testing the safety of an AD vaccine, since even AN1792 did not induce inflammation in various types of animals, including APP transgenic mouse models of AD. In addition, I think it is unlikely that the FDA or other regulatory agencies will allow another clinical trial based on full-length Aβ42 peptide immunization.

Another important issue is the plasmid that has been delivered by a gene-gun device approved only for animal use. The authors of this paper suggest that the double plasmid system (DPS) used in this study could enhance Aβ42 expression and trigger stronger immunity. However, as presented in the text, the authors generated only ~15 μg/ml antibodies (exact titers, individual variability and SD are not presented); this is much lower than the antibody titers (500 μg/ml) detected in mice vaccinated with Aβ42 formulated in Quil A adjuvant. Incidentally, using a single and effective pCMVE plasmid encoding an epitope AD vaccine we generated ~1,000 μg/ml and ~450 μg/ml of antibodies in wild-type and APP/Tg mice of H2b haplotypes, respectively (3). Thus, using such a complicated plasmid system is not necessary to generate a strong immune response. To assess the safety of this DPS, I respectfully submit that one should check the antibody and T cell responses not only to amyloid, but also to the GAL4 transcriptional factor. This foreign protein could be exposed to B cells from apoptotic cells transfected with DPS and almost certainly will be presented through MHC class I and II to the immune system of the host. In this experiment the host was the B6SJL strain of mice, but in case of a clinical trial it would be a human population with high MHC polymorphism.

Finally, the authors of this study are claiming that their DNA vaccine is inducing only the Th2-type of immune responses. It is likely that when using full-length Aβ42, one should aim to avoid pro-inflammatory (Th1) immune responses and induce a Th2 polarized anti-inflammatory response. The authors used an indirect measure of Th responses, analyzing Ig isotypes after AD vaccination as we suggested previously (6). Next, the authors rightly decided to measure cytokine production in splenocyte cultures from mice immunized with DNA or peptide. Unfortunately, the data of these experiments are presented only in the text of the manuscript in such a way that it is extremely difficult to understand the results of this study. There are no graphs, statistical analyses, and information about control groups of mice non-immunized, or immunized with irrelevant antigen. Without such clearly presented data, it is impossible to understand the immunogenic potency of DPS expressing full-length amyloid.

In conclusion, I would like to mention that a very recent assessment of the relationship between Aβ42 immune responses, degree of plaque removal, and long-term clinical outcomes demonstrated that immunization with Aβ42 resulted in clearance of amyloid plaques in the brains of AD patients, but did not prevent progressive neurodegeneration. These data suggest that removal of existing plaques alone is not sufficient to stop the progression of neurodegeneration and improve cognitive function. Another potential problem of AD immunotherapy in general could be the fact that a reduction of insoluble Aβ (plaques) may lead to increased levels of soluble forms of this peptide, primarily oligomers, the most toxic form for neurons, and impair cognitive function. Thus, I believe that to avoid these problems one should generate an AD vaccine that could be administered before significant AD brain pathology has accumulated. Passive transfer of anti-Aβ42 antibodies may be impractical for long-term preventative/early therapeutic application in AD because of the requirement for frequent intravenous dosing and the high cost of treatment. Thus, an ideal AD vaccine should be, first of all, extremely safe and, secondly, highly effective so it can be used prophylactically in high-risk subjects or therapeutically in early-stage AD. Unfortunately, this paper does not address these important questions.

References:
1. Ghochikyan A, Vasilevko V, Petrushina I, Movsesyan N, Babikyan D, Tian W, Sadzikava N, Ross TM, Head E, Cribbs DH, Agadjanyan MG. Generation and characterization of the humoral immune response to DNA immunization with a chimeric beta-amyloid-interleukin-4 minigene. Eur J Immunol. 2003 Dec;33(12):3232-41. Abstract

2. Davtyan H, Mkrtichyan M, Movsesyan N, Petrushina I, Mamikonyan G, Cribbs DH, Agadjanyan MG, Ghochikyan A. DNA prime-protein boost increased the titer, avidity and persistence of anti-Abeta antibodies in wild-type mice. Gene Ther. 2009 Oct 29. Abstract

3. Movsesyan N, Ghochikyan A, Mkrtichyan M, Petrushina I, Davtyan H, Olkhanud PB, Head E, Biragyn A, Cribbs DH, Agadjanyan MG. Reducing AD-like pathology in 3xTg-AD mouse model by DNA epitope vaccine - a novel immunotherapeutic strategy. PLoS One. 2008;3(5):e2124. Abstract

4. Movsesyan N, Mkrtichyan M, Petrushina I, Ross TM, Cribbs DH, Agadjanyan MG, Ghochikyan A. DNA epitope vaccine containing complement component C3d enhances anti-amyloid-beta antibody production and polarizes the immune response towards a Th2 phenotype. J Neuroimmunol. 2008 Dec 15;205(1-2):57-63. Abstract

5. Movsesyan, N. et al. Alzheimer's Disease DNA epitope vaccine induces equally strong humoral immune responses after delivery both via electroporation and gene gun. In prep.

6. Cribbs DH, Ghochikyan A, Vasilevko V, Tran M, Petrushina I, Sadzikava N, Babikyan D, Kesslak P, Kieber-Emmons T, Cotman CW, Agadjanyan MG. Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid. Int Immunol. 2003 Apr;15(4):505-14. Abstract

View all comments by Michael G. Agadjanyan

We appreciate the comment of Dr. Agadjanyan. We are aware of the work done by his group in the field of Aβ immunizations and recognize this in references 31 to 33.

Our manuscript underwent the regular peer review process and was deemed sufficiently innovative for publication. A novel approach in DNA vaccinations was applied using a double plasmid system with which we achieved not high, but effective antibody levels resulting from a non-inflammatory Th2 polarized immune response. One of the authors, Roger Rosenberg, as disclosed in the paper, is a co-inventor of a US patent application for “Aβ gene vaccines”. The fact that a patent was obtained is another indicator that our approach is innovative and relevant. Our group has made published contributions to this field of research since 2003 and we have presented the double plasmid approach at the ICAD meetings in 2008 and 2009.

The multiple copies of Aβ are linked together, making a trimeric protein rather than just expressing the single peptide three times. This is the most novel part of the paper in terms of a vaccine....  Read more

We appreciate the comment of Dr. Agadjanyan. We are aware of the work done by his group in the field of Aβ immunizations and recognize this in references 31 to 33.

Our manuscript underwent the regular peer review process and was deemed sufficiently innovative for publication. A novel approach in DNA vaccinations was applied using a double plasmid system with which we achieved not high, but effective antibody levels resulting from a non-inflammatory Th2 polarized immune response. One of the authors, Roger Rosenberg, as disclosed in the paper, is a co-inventor of a US patent application for “Aβ gene vaccines”. The fact that a patent was obtained is another indicator that our approach is innovative and relevant. Our group has made published contributions to this field of research since 2003 and we have presented the double plasmid approach at the ICAD meetings in 2008 and 2009.

The multiple copies of Aβ are linked together, making a trimeric protein rather than just expressing the single peptide three times. This is the most novel part of the paper in terms of a vaccine. Because B cells recognize antigens in solution and require receptor crosslinking for activation, multimeric epitopes are able to elicit B cell responses more potently than single epitopes. The double plasmid system, in which one plasmid encodes a yeast transcription factor, which drives the transcription of the Aβ42 sequence on the second plasmid, resulted in an about 10-fold stronger antibody production and improves the DNA Aβ42 trimer vaccine substantially. It is still much less compared to the peptide-immunized mice, but as we stated in the paper “a predominant Th2 response is a more important objective in developing an effective and safe therapy for AD than increased antibody levels alone”. It must be noted that T cell help is obviously provided in this model because isotype switching has occurred.

A major finding of this paper is that despite multiple immunizations T cell responses are undetectable. T cell responses are likely to be the cause of the encephalitis found in the clinical trial of peptide vaccination.

As stated by Dr. Agadjanyan, Aβ42 is a T cell-dependent antigen. Is it safe to generate a T and a B cell response by a DNA vaccine encoding full-length Aβ42? First, to have an effective immune response, both T and B lymphocytes are needed, as both strongly influence each other. In our manuscript, we do not state that our vaccine does not activate T cells. Instead, we say that T cell have been involved in the early immunizations process, as it is clearly indicated by the isotype switch of the respective Aβ42 antibodies produced. However, we could not detect in-vitro T cell activation or proliferation against Aβ42 as documented by appropriate detection assays. Since we did not find a prolonged T cell response, we conclude this type of vaccine is probably safe for use in humans. We are currently performing experiments to characterize early T cell responses and to determine T cell fate longitudinally. Second, the exact B and T cell epitopes in humans have not yet been clearly defined, as most of the work to date is done in mouse strains. Thus, we currently have insufficient data to avoid certain components of the Aβ42 peptide (for example: the T cell epitopes in the mouse) in the development of an effective vaccine for human AD patients.

Sufficient statistical analyses are provided in our manuscript. Every data point consists of mean and standard deviation. Additional information on the number of animals and the statistical assessment are provided in the article.

We agree with Dr. Agadjanyan that there is ongoing scientific discussion regarding the relevance of the plaque hypothesis. Removal of established plaques did not ameliorate or prevent progression of the disease in one clinical trial. Although the plaque count was reduced dramatically in these patients, there was evidence of progressive cognitive decline (Holmes et al., 2008). While it is very challenging to study these two seemingly contradictory observations in human patients, intuitively one would speculate that therapy was initiated too late when irreversible progressive and independent pathologic changes had already occurred. The greatest challenge for investigators who attempt to prevent clinical and pathological aspects of AD by DNA immunization or other interventional means will be the identification of biomarkers that will allow an early therapy. In summary, we think that our manuscript addresses a number of important key questions and we welcome further discussion.

References:
Holmes C, Boche D, Wilkinson D, Yadegarfar G, Hopkins V, Bayer A, Jones RW, Bullock R, Love S, Neal JW, Zotova E, Nicoll JA. Long-term effects of Abeta42 immunization in Alzheimer's disease: follow-up of a randomized, placebo-controlled phase I trial. Lancet. 2008; 372:216-223. Abstract

View all comments by Doris Lambracht-Washington
View all comments by Roger N. Rosenberg

The development of innovative vaccination strategies for Alzheimer disease is of utmost importance. In this article, a novel and interesting DNA-vaccine approach with two plasmids is described. Unfortunately, the antibody response raised in the non-transgenic mice is still fairly low, with OD-values ~0.5 when plasma samples are diluted only 1:500 (Fig. 3). One of the main difficulties with a DNA vaccine is to raise a significant immune response in humans or primates, but this problem is not addressed. The description of experimental procedures lack important details and their findings are presented in a somewhat incomplete manner. For instance, in Fig. 4, splenocytes from only a few mice were restimulated, and then only with the Aβ42 peptide. The authors conclude that a T cell response is undetectable. The obvious control experiment would have been to restimulate with the trimeric Aββ42 peptide. It is unclear if the splenocyte cultures contained only T cells, or were a mixture of T and B cells. If so, it would have been interesting to investigate if the isotype profile of the...  Read more

The development of innovative vaccination strategies for Alzheimer disease is of utmost importance. In this article, a novel and interesting DNA-vaccine approach with two plasmids is described. Unfortunately, the antibody response raised in the non-transgenic mice is still fairly low, with OD-values ~0.5 when plasma samples are diluted only 1:500 (Fig. 3). One of the main difficulties with a DNA vaccine is to raise a significant immune response in humans or primates, but this problem is not addressed. The description of experimental procedures lack important details and their findings are presented in a somewhat incomplete manner. For instance, in Fig. 4, splenocytes from only a few mice were restimulated, and then only with the Aβ42 peptide. The authors conclude that a T cell response is undetectable. The obvious control experiment would have been to restimulate with the trimeric Aββ42 peptide. It is unclear if the splenocyte cultures contained only T cells, or were a mixture of T and B cells. If so, it would have been interesting to investigate if the isotype profile of the humoral response in blood samples (in Fig. 2) could have been replicated when cultured cells were restimulated. We regard this article as potentially interesting, but still very preliminary. Efficacy and safety experiments in transgenic mice and/or primates will be needed to evaluate this novel DNA vaccine strategy before it can be clinically tested.

View all comments by Frida Ekholm Pettersson
View all comments by Lars Nilsson

I'd like to thank Dr. Lambracht-Washington et al. for their reply and respond to a few points.

Patents for specific plasmids are fairly routinely granted. Such a patent is not in and of itself proof of scientific innovation on the present challenges of Alzheimer disease vaccine design. The T cell epitope of amyloid in humans has been reported by Monsonego et al. (1); the B cell epitope in humans is very well described by the Elan group (2), and our group has characterized the mouse T cell epitope (3).

I agree with the authors' claim that the novel part of their study would be the trimeric protein. If so, the study would be well advised to focus its data and discussion around this new aspect. However, the paper contains no data characterizing the form of peptide expressed from their plasmid. There are no data to show that multiple copies of Aβ are expressed linked together into a trimer rather than three times as single peptides. The presumed trimeric antigen is not characterized.

References:
1. Monsonego A, Zota V, Karni A, Krieger JI, Bar-Or A, Bitan G, Budson AE, Sperling R, Selkoe DJ, Weiner HL. Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease. J Clin Invest. 2003 Aug;112(3):415-22. Abstract

2. Lee M, Bard F, Johnson-Wood K, Lee C, Hu K, Griffith SG, Black RS, Schenk D, Seubert P. Abeta42 immunization in Alzheimer's disease generates Abeta N-terminal antibodies. Ann Neurol. 2005 Sep;58(3):430-5. Abstract

3. Cribbs DH, Ghochikyan A, Vasilevko V, Tran M, Petrushina I, Sadzikava N, Babikyan D, Kesslak P, Kieber-Emmons T, Cotman CW, Agadjanyan MG. Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid. Int Immunol. 2003 Apr;15(4):505-14. Abstract

View all comments by Michael G. Agadjanyan

The study by Lambracht-Washington and coworkers explores the possibility of using a DNA-based Aβ1-42 trimer vaccine to promote Th2-type, Aβ1-42-specific immune response in wild-type mice. As detailed above in comments from Michael Agadjanyan, this work extends from pioneering studies that were previously conducted in the area of DNA-based vaccination against Alzheimer’s. Those past studies, a number of which were conducted in the Agadjanyan and Cribbs labs, definitively established that DNA-based Aβ vaccines were efficacious in reducing cerebral amyloidosis in transgenic Alzheimer’s mouse models (for a review, see Town, 2009). I agree with Michael that the novelty in the present study is primarily owing to use of a trimeric Aβ1-42 vaccine administered via a gene-gun delivery system, and secondarily, due to utilization of a dual plasmid system that relies on GAL4 transactivation of the 12.2 kD Aβ1-42 trimer antigen.

I would like to add a few comments regarding this manuscript. It seems that the amount of anti-Aβ1-42 antibodies is rather low, even after a prime-boost regimen of...  Read more

The study by Lambracht-Washington and coworkers explores the possibility of using a DNA-based Aβ1-42 trimer vaccine to promote Th2-type, Aβ1-42-specific immune response in wild-type mice. As detailed above in comments from Michael Agadjanyan, this work extends from pioneering studies that were previously conducted in the area of DNA-based vaccination against Alzheimer’s. Those past studies, a number of which were conducted in the Agadjanyan and Cribbs labs, definitively established that DNA-based Aβ vaccines were efficacious in reducing cerebral amyloidosis in transgenic Alzheimer’s mouse models (for a review, see Town, 2009). I agree with Michael that the novelty in the present study is primarily owing to use of a trimeric Aβ1-42 vaccine administered via a gene-gun delivery system, and secondarily, due to utilization of a dual plasmid system that relies on GAL4 transactivation of the 12.2 kD Aβ1-42 trimer antigen.

I would like to add a few comments regarding this manuscript. It seems that the amount of anti-Aβ1-42 antibodies is rather low, even after a prime-boost regimen of up to eight total DNA vaccinations. For instance, it appears that DNA-vaccinated mice produced ~fivefold less antibodies against full-length anti-Aβ1-42 versus peptide-vaccinated mice (and that is a conservative estimate assuming linearity of OD readings for antibody titres; see Fig. 3) and ~five- to sixfold reduced stimulation of T cell proliferation following Aβ1-42 splenocyte challenge compared to peptide immunization (see Fig. 4). Because the authors only chose to examine wild-type mice, it is unclear whether this relatively weak Aβ1-42 antibody response would be efficacious, and thereby translate to reduced cerebral amyloidosis in mice, let alone in humans. It is possible that the trimeric Aβ1-42 antigen is not optimal for generating antibody responses to synthetic Aβ1-42; however, the authors do not seem to report results from trimeric Aβ1-42 splenocyte recall stimulation experiments, which may have resulted in stronger anti-Aβ antibody responses.

I’d like to build on a comment that Michael made regarding assessment of Aβ immunotherapy safety. In the “Comment” section of this article, the authors state that they can assess safety of the DNA-based approach by comparing immune responses between DNA-based trimeric vaccination and peptide-based immunization conducted side-by-side. This is flawed logic. With the exception of one unconfirmed report that utilized pertussis toxin in combination with “standard” Aβ immunotherapy using Aβ1-42 plus CFA/IFA (Furlan et al., 2003), none of the Aβ immunotherapy approaches have produced aseptic meningoencephalitis—nor any other severe adverse event—in mice. Because of this, it is not possible to make any conclusions about safety by simply assessing Th1 or Th2 immune responses after DNA Aβ1-42 trimer vaccination in mice.

Finally, I would like to call the authors’ attention to our previous work, which was one of the first demonstrations that “standard” Aβ1-42 immunotherapy (as originally developed by Schenk et al., 1999) primarily produces a Th2 response in mice as determined by 1) primarily IgG1 anti-Aβ1-42 antibodies that recognized amino acids 1-12 of the peptide (now widely regarded as the B cell epitope), and 2) primarily Th2 cytokines both in vivo in mouse plasma and ex vivo in splenocyte assays (Town et al., 2001; Town et al., 2002). More recently, we have shown that changing the route of vaccination to transcutaneous delivery results in copious amounts of anti-Aβ antibodies, a primarily Th2-type immune response, and clearance of cerebral amyloid (Nikolic et al., 2007). Thus, it is possible to generate Th2 immune responses in mice using simpler, peptide-based approaches to Aβ immunotherapy. The key challenges are to translate these approaches into the clinic by devising strategies that are both safe and efficacious.

References:
Furlan R, Brambilla E, Sanvito F, Roccatagliata L, Olivieri S, Bergami A, Pluchino S, Uccelli A, Comi G, Martino G. Vaccination with amyloid-beta peptide induces autoimmune encephalomyelitis in C57/BL6 mice. Brain. 2003 Feb;126(Pt 2):285-91. Abstract

Nikolic WV, Bai Y, Obregon D, Hou H, Mori T, Zeng J, Ehrhart J, Shytle RD, Giunta B, Morgan D, Town T, Tan J. Transcutaneous beta-amyloid immunization reduces cerebral beta-amyloid deposits without T cell infiltration and microhemorrhage. Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2507-12. Abstract

Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D, Seubert P. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 1999 Jul 8;400(6740):173-7. Abstract

Town T. Alternative Abeta immunotherapy approaches for Alzheimer's disease. CNS Neurol Disord Drug Targets. 2009 Apr;8(2):114-27. Abstract

Town T, Tan J, Sansone N, Obregon D, Klein T, Mullan M. Characterization of murine immunoglobulin G antibodies against human amyloid-beta1-42. Neurosci Lett. 2001 Jul 13;307(2):101-4. Abstract

Town T, Vendrame M, Patel A, Poetter D, DelleDonne A, Mori T, Smeed R, Crawford F, Klein T, Tan J, Mullan M. Reduced Th1 and enhanced Th2 immunity after immunization with Alzheimer's beta-amyloid(1-42). J Neuroimmunol. 2002 Nov;132(1-2):49-59. Abstract

View all comments by Terrence Town

Clearly, more work needs to be accomplished to define the immune response with Aβ42 immunization in both experimental animal models and in patients, but we are dedicated to proceeding in this direction.

Two related hypotheses drive our research: First, the generation of an effective anti-Aβ42 B cell response that will result in an antibody-mediated clearance of amyloid plaques, leading to a reduction in AD severity. Second, from a bio-safety standpoint, the generation of an anti-Aβ42 Th1 effector autoimmune response may lead to immune mediated pathology. These hypotheses are based on research by many others, including the clinical trial using peptide vaccination. We have sought to develop a therapy that will generate an effective antibody response and will lack measurable Aβ42-specific Th1 T cell responses. At this point, the definition of "effective" may not be directly related to Ig titer but is rather a biological outcome-based definition, as we have detected clearance of plaque with low titers previously and there is no method currently available to test CSF or CNS titers...  Read more

Clearly, more work needs to be accomplished to define the immune response with Aβ42 immunization in both experimental animal models and in patients, but we are dedicated to proceeding in this direction.

Two related hypotheses drive our research: First, the generation of an effective anti-Aβ42 B cell response that will result in an antibody-mediated clearance of amyloid plaques, leading to a reduction in AD severity. Second, from a bio-safety standpoint, the generation of an anti-Aβ42 Th1 effector autoimmune response may lead to immune mediated pathology. These hypotheses are based on research by many others, including the clinical trial using peptide vaccination. We have sought to develop a therapy that will generate an effective antibody response and will lack measurable Aβ42-specific Th1 T cell responses. At this point, the definition of "effective" may not be directly related to Ig titer but is rather a biological outcome-based definition, as we have detected clearance of plaque with low titers previously and there is no method currently available to test CSF or CNS titers which are more relevant than serum (1,2). We specifically chose to test these hypotheses in non-transgenic mice in order to provide the most stringent possible test for T cell responses, as these mice have no thymic negative selection against human Aβ42. Therefore, if the potential existed for the development of a Th1 response, it should be detectable in this model (null hypothesis). Therefore, we compared the immune responses in wild-type mice against DNA and Aβ42 peptide immunization.

This direct comparison is innovative and constructive, as the results show a clearly different immune response with a predominant Th2 immune response with DNA Aβ42 immunization (Th2/Th1 ratio 10) and a mixed immune response with Aβ42 peptide (Th2/Th1 ratio 1). Furthermore, T cell proliferation (as determined by the stimulation indices) following DNA versus peptide immunizations showed again very different outcomes with non-reactive T cells after re-stimulation with Aβ42 peptide after DNA Aβ42 immunization and reactive T cells after Aβ42 peptide immunization.

These are important findings and strongly support proceeding ahead with a clinical trial using DNA Aβ42 immunization in patients. Additional studies using this construct in APP transgenic mice are ongoing as well as further characterization of the trimeric Aβ42 peptide as the new and innovative antigen in our system. We are aware of the challenge that the outcome in humans may or may not be the same as the findings in mice, but our study offers a valid explanation as to why the clinical trial with Aβ42 peptide immunization had caused encephalitis in 6 percent of the patients. We agree with Terrence Town, that “none of the Aβ immunotherapy approaches have produced aseptic meningoencephalitis nor any other severe adverse event in mice,” but our data are in strong support that DNA Aβ42 immunization does offer a potentially lower risk for adverse effects in patients. Because the meningoencephalitis was likely due an inflammatory Th1 immune response caused by the type of adjuvant used, we agree that it is possible to generate an non-inflammatory immune response using simpler, peptide-based approaches to Aβ immunotherapy, but we would like to cite a statement by Michael Agadjanyan in the first comment to our paper: “scale-up of manufacturing for clinical trials is readily achieved, and cGMP-grade DNA vaccines are significantly less expensive than peptide-based cGMP vaccines.”

These discussions with our colleagues are constructive and help to provide focus for future research.

References:
1. Qu B, Boyer PJ, Johnston SA, Hynan LS, Rosenberg RN. Abeta42 gene vaccination reduces brain amyloid plaque burden in transgenic mice. J Neurol Sci. 2006; 244:151-158. Abstract

2. Qu B-X, Xiang Q, Li L, Johnston SA, Hynan LS, Rosenberg RN. Abeta42 gene vaccine prevents Abeta42 deposition in brain of double transgenic mice J Neurol Sci. 2007; 260: 204-213. Abstract

View all comments by Doris Lambracht-Washington

I’d like to add a few points to this discussion. I agree that the double plasmid system with GLA4 Activator, UAS/AB42 Trimer responder is quite innovative; however, whether this approach has translational potential for a human clinical trial is doubtful based on the data provided in the current manuscript.

I found it interesting that the wild-type mice used in this study were female B6SJLF1/J mice, which is the background of the Tg2576 mice that show immune hypo-responsiveness to immunization with Aβ42 (1). No data were provided in the current manuscript using APP transgenic mice to show that the antibody response induced by the double plasmid system was capable of attenuating amyloid deposition or improving behavioral measures. This raises the issue of immune tolerance that needs to be broken in order to induce an adequate antibody response to a “self” peptide or protein. In addition, because the target population for anti-Aβ immunotherapy is the elderly, overcoming immunosenescence is a formidable hurdle as well. The generally low titers and the low number of positive...  Read more

I’d like to add a few points to this discussion. I agree that the double plasmid system with GLA4 Activator, UAS/AB42 Trimer responder is quite innovative; however, whether this approach has translational potential for a human clinical trial is doubtful based on the data provided in the current manuscript.

I found it interesting that the wild-type mice used in this study were female B6SJLF1/J mice, which is the background of the Tg2576 mice that show immune hypo-responsiveness to immunization with Aβ42 (1). No data were provided in the current manuscript using APP transgenic mice to show that the antibody response induced by the double plasmid system was capable of attenuating amyloid deposition or improving behavioral measures. This raises the issue of immune tolerance that needs to be broken in order to induce an adequate antibody response to a “self” peptide or protein. In addition, because the target population for anti-Aβ immunotherapy is the elderly, overcoming immunosenescence is a formidable hurdle as well. The generally low titers and the low number of positive responders in the AN1792 clinical trial (I say that with the proviso that the trial was halted before the patients completed the protocol) illustrate the difficulties facing future active immunization trials in this elderly patient population. Therefore, inducing robust anti-Aβ antibody responses in APP transgenic mouse models, as well as large animal models, will likely be a prerequisite before moving forward into AD patients. In fact, we have previously published a study in elderly dogs that used fibrillar Aβ42 and alum as a Th2 adjuvant approved for human use; this induced therapeutic levels of anti-Aβ antibody titers, sufficient to clear amyloid deposits, in all of the immunized canines (2).

Other commentators on Alzforum have already pointed out the large number of immunizations required to induce rather low titers of anti-Aβ antibodies in this study, as well as the significant potential to induce an anti-GLA4 protein immune response. The latter would likely result in the loss of cells expressing the GLA4 protein, thereby eliminating the expression of the Aβ42 trimer immunogen. I would like to emphasize the fact that just because you fail to measure a T cell response from immunized mice does not mean that there was no response. Typically a T cell response is required to get Ig class switching from IgM to the IgG isotypes; Lambracht-Washington et al. were able to measure IgG1 and IgG2a isotypes, thus indicating that there was a T cell response to the Aβ42 trimer immunogen. Thus, the problem was likely due to the weak T cell response that was induced with the double plasmid system, thereby making it difficult to detect in the re-stimulated splenocyte cultures, which was also reflected in the rather weak antibody response to the double plasmid system.

A second major problem is the large number of immunizations needed to get the anti-Aβ antibody titers to 15 ug/ml. In fact, the data in Figure 3 do not appear to show any difference between the mice (n = 4) that received six immunizations and those (n = 4) that received 14 immunizations if you compare the antibody binding in panels A and B, because all of the plasma samples were diluted 1:500. Multiple immunization protocols—especially DNA immunization protocols requiring 6-14 injections that also require specialized equipment, such as electroporation, to promote adequate expression levels of the antigen to induce therapeutic levels of antibodies—are probably not a viable approach for translation to human clinical trials. Therefore, inclusion of some sort of adjuvant in the vaccine design to enhance the antibody titers and reduce the number of immunizations, either as a fusion construct or as a supplement (co-injection or transcutaneous delivery) to the immunogen will be required.

References:
1. Monsonego A, Maron R, Zota V, Selkoe DJ, Weiner HL. Immune hyporesponsiveness to amyloid beta-peptide in amyloid precursor protein transgenic mice: implications for the pathogenesis and treatment of Alzheimer's disease. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10273-8. Epub 2001 Aug 21. Abstract

2. Head E, Pop V, Vasilevko V, Hill M, Saing T, Sarsoza F, Nistor M, Christie LA, Milton S, Glabe C, Barrett E, Cribbs D. A two-year study with fibrillar beta-amyloid (Abeta) immunization in aged canines: effects on cognitive function and brain Abeta. J Neurosci. 2008 Apr 2;28(14):3555-66. Abstract

View all comments by David Cribbs

We thank David Cribbs for his comment, and we would like to correct a few things. First of all, we did not show the results from 14 immunizations in our mice. We immunized one group receiving a total of six DNA immunizations and a second group which received a total of eight DNA Aβ42 trimer immunizations. The resulting antibody titers are 10-fold higher than antibody titers from a previous published immunization construct from our group which was an Aβ42 monomer. For immunizations with the monomer, we have shown an effective reduction in plaque load in an APP transgenic mouse model. Therefore, we conclude that the new DNA Aβ42 trimer is very likely to cause an even better or at least equal reduction in overall plaque load and is thus effective and sufficient to cause a therapeutic effect. Further studies using DNA Aβ42 trimer immunization in transgenic mouse models are underway. Again, we would like to emphasize that an initial T cell response was observed by the isotype switching of the respective Aβ42 specific antibodies, but then the T cell response disappeared, not due to...  Read more

We thank David Cribbs for his comment, and we would like to correct a few things. First of all, we did not show the results from 14 immunizations in our mice. We immunized one group receiving a total of six DNA immunizations and a second group which received a total of eight DNA Aβ42 trimer immunizations. The resulting antibody titers are 10-fold higher than antibody titers from a previous published immunization construct from our group which was an Aβ42 monomer. For immunizations with the monomer, we have shown an effective reduction in plaque load in an APP transgenic mouse model. Therefore, we conclude that the new DNA Aβ42 trimer is very likely to cause an even better or at least equal reduction in overall plaque load and is thus effective and sufficient to cause a therapeutic effect. Further studies using DNA Aβ42 trimer immunization in transgenic mouse models are underway. Again, we would like to emphasize that an initial T cell response was observed by the isotype switching of the respective Aβ42 specific antibodies, but then the T cell response disappeared, not due to weak T cell response but due to a Th2 response which is non-inflammatory. The reason why we choose B6SJLF1 mice was indeed because this strain is the genetic background for a number of APP transgenic mouse strains. B6SJLF1 is a good responder in Aβ42 immunizations, as it carries the mixed background of C57BL6 which is poorly responding to Aβ42 immunization but has a genetic background promoting brain plaque development and SJL which has a great Aβ42 immune response.

View all comments by Doris Lambracht-Washington

I would like to respond to a reply made above by Doris Lambracht-Washington concerning the Th2 response issue raised by David Cribbs. Her reply was “Again, we would like to emphasize that an initial T cell response was observed by the isotype switching of the respective Aβ42 specific antibodies, but then the T cell response disappeared, not due to weak T cell response but due to a Th2 response which is non-inflammatory.”

The type of T cell response, i.e., Th1 vs. Th2, is not defined in any way by the duration or persistence of the response. These Th profiles are defined based on the types of cytokines produced; e.g., Th1 responses are primarily associated with interferon-gamma and interleukin-12, whereas Th2 responses occur with interleukin-4 and interleukin-10 production, amongst others such as interleukin-15. Thus, the Th2 response to trimeric Aβ antigen observed by Lambracht-Washington and coworkers would not, by its very nature, have a limited duration because it is anti-inflammatory.

Furthermore, it is a misconception that Th2 responses are always...  Read more

I would like to respond to a reply made above by Doris Lambracht-Washington concerning the Th2 response issue raised by David Cribbs. Her reply was “Again, we would like to emphasize that an initial T cell response was observed by the isotype switching of the respective Aβ42 specific antibodies, but then the T cell response disappeared, not due to weak T cell response but due to a Th2 response which is non-inflammatory.”

The type of T cell response, i.e., Th1 vs. Th2, is not defined in any way by the duration or persistence of the response. These Th profiles are defined based on the types of cytokines produced; e.g., Th1 responses are primarily associated with interferon-gamma and interleukin-12, whereas Th2 responses occur with interleukin-4 and interleukin-10 production, amongst others such as interleukin-15. Thus, the Th2 response to trimeric Aβ antigen observed by Lambracht-Washington and coworkers would not, by its very nature, have a limited duration because it is anti-inflammatory.

Furthermore, it is a misconception that Th2 responses are always “non-inflammatory.” A number of immune disorders, including allergy and atopic disorders such as asthma are driven by overly aggressive Th2 responses.

View all comments by Terrence Town