De Novo Design of Peptide Immunogens

My journal club review of the paper entitled "De Novo Design of Peptide Immunogens That Mimic the Coiled Coil Region of Human T-cell Leukemia Virus Type-1 Glycoprotein 21 Transmembrane Subunit for Induction of Native Protein Reactive Neutralizing Antibodies" by Roshni Sundaram, Marcus P. Lynch, Sharad V. Rawale, Yiping Sun, Mirdad Kazanji, and Pravin T. P. Kaumaya, published in The Journal of Biological Chemistry (Vol. 279, No. 23, pp. 24141–24151), in the year 2004.

==Overview==

Human T-cell Leukemia virus type-1 (HTLV-1) is a considerable medical problem across the globe and there is lack of effective therapy for HTLV-1 associated diseases. Vaccination, therefore, represents an ideal approach to control the virus. In this study, Sundaram et al., assessed the potential of two de novo designed peptide immunogens (WCCR2T, wild-type peptide; CCR2T, leucine-substituted peptide) that mimicked the coiled coil region of HTLV-1 glycoprotein (gp21) transmembrane (TM) subunit for induction of neutralizing antibodies against the virus. Their novel design involved utilizing a beta-sheet template backbone consisting of alternating Gly/Lys residues that allowed growth of peptide immunogens onto the epsilon side chains of three lysine residues, potentiating the peptides to form triple helical coiled-coil conformation, mimicking the native structure. The template backbone was linked to a “promiscuous” helper T-cell epitope from tetanus toxin to overcome the heterogeneity of the major histocompatibility complex (MHC) in mice population and provide boost to the immune response. Their results showed that antibodies generated using both peptide immunogens were directed against the coiled-coil region of the native gp21 envelope glycoprotein and were neutralizing as they resulted in reduction of cell-cell fusion. These results validated the authors’ hypothesis, demonstrated the validity of their template design and indicated that the two peptide immunogens represent potential candidates for use in a peptide vaccine against HTLV-1. The data presented in this study and the insights gained represent an important step forward towards the design of a vaccine effective against HTLV-1. Further, the approach presented in this paper can be used as a template for the design of peptide vaccines able to induce high affinity neutralizing antibody responses for other pathogens, such as HIV (Louis et al., 2003), SARS-CoV (Ingallinella et al., 2004) and influenza virus (Skehel and Wiley, 2000), which also utilize coiled coil conformation for the fusion process. Additionally, antibodies produced against the immunogens are of significant utility for the analysis of TM function in the respective pathogen infections.

The authors should be praised for taking extensive measures to ensure the generation of structurally and functionally viable peptide immunogen constructs. This was necessary to circumvent the several limitations of synthetic peptide vaccines (Bona et al., 1998), such as low immunogenicity, chemical and conformational instability, short serum half-life, rapid degradation by proteolytic enzymes, and restrictions posed by MHC diversity, among others.

Despite the validation of the hypothesis, the thorough work performed, there are several major and minor deficiencies in their paper, which I highlight below.

==Major points==

1) Page 24144: Ideally, strategies to design protective vaccines must be able activate the relevant arm(s) of the immune system, in particular the humoral and the cellular immune responses. The addition of promiscuous helper T-cell epitope to the template peptide immunogen was, therefore, impeccable, as it will not only help trigger cellular immune response but also help overcome the polymorphism of MHC molecules. However, the rationale for selection of tetanus toxoid peptide was not well justified as it is only good for priming and the resulting memory T helper cells are not likely to be helpful upon re-infection by HTLV-1. Why did the authors not consider using a promiscuous helper epitope from the HTLV-1 virus itself?

2) Page 24146 and 24148: It is possible that the CCR2T and WCCR2T antisera used for ELISA assays (Fig. 5 and Fig. 8) also contain antibodies that bind to the promiscuous T-cell epitope on the peptide immunogen. So, in addition to measuring antibodies binding specifically to the conformational epitope, the authors could also be measuring antibodies binding to the promiscuous T-cell epitope. Also, if this was true, antibodies binding to the B- and T-cell epitopes could lead to steric hindrances in binding analysis when the immunogen is immobilized onto ELISA plates. Hence, considering these possibilities, the binding and titers observed by the authors may not be an accurate representation of actual affinity for the native protein.

3) Page 24149: The authors showed that their peptide immunogens are neutralizing in that they are capable of disrupting cell-cell fusion. However, it should be noted that neutralizing antibodies are not necessarily protective (Lairmore et al., 1995). A challenge study is, therefore, necessary to assess whether the peptide immunogens are protective. The authors do not mention this limitation in their discussion.

4) Page 24143. Mice, which are not hosts for HTLV-1 infection, were used for immunization and the resulting sera was used for majority of the assays performed; it is, therefore, not clear how well the mice data will correspond to data from human. Over the years, many animal models of HTLV-1 infection have been developed for the purpose of testing various vaccine candidates, such as rabbits (Lairmore et al., 1992) and squirrel monkey, Saimiri sciureus (Kazanji, 2000). The results obtained by the authors would have been more relevant had they chosen these animals for their study.

5) Page 24141: The authors said that their results suggest that the two peptide immunogens represent potential candidates for future multivalent vaccine studies. It should be noted that despite the advantages of short synthetic peptides as vaccines, only a small percentage of such peptides, evaluated in preclinical trials, have progressed to clinical trials (Hans et al., 2006). Though the authors’ initial results with the peptide immunogens in mice are promising, it remains to be seen how well the immunogens fare in the subsequent evaluation stages.

6) Page 24146: In figure 5, the pre-serum values, representing negative control, should be included.

==Minor points==

1) Page 24144: The position numbers in “V349L, I353L, I360L, N363L, and I370L” do not correspond to the B-cell epitope peptide sequence provided in Table 1.
2) Page 24144: The period (.) between the sentences “….are indicated by an asterisk” and “CCR2E and WCCR2E are the…” is missing.
3) Page 24146: “background” and not “backround”.
4) Page 24141: “B-cell epitope” and not “B cell epitope”.
5) Page 24142, 24143 and 24150: Inconsistency with the phrase “coiled coil”, majority of the times it is written as “coiled coil” and sometimes as “coiled-coil”.

==References==

Kazanji M. HTLV type 1 infection in squirrel monkeys (Saimiri sciureus): a promising animal model for HTLV type 1 human infection. AIDS Res Hum Retroviruses. 2000 Nov 1;16(16):1741-6.

Lairmore MD, Rudolph DL, Roberts BD, Dezzutti CS, Lal RB. Characterization of a B-cell immunodominant epitope of human T-lymphotropic virus type 1 (HTLV-I) envelope gp46. Cancer Lett. 1992 Sep 14;66(1):11-20.

Lairmore MD, DiGeorge AM, Conrad SF, Trevino AV, Lal RB, Kaumaya PT. Human T-lymphotropic virus type 1 peptides in chimeric and multivalent constructs with promiscuous T-cell epitopes enhance immunogenicity and overcome genetic restriction. J Virol. 1995 Oct;69(10):6077-89.

Hans D, Young PR, Fairlie DP. Current status of short synthetic peptides as vaccines. Med Chem. 2006 Nov;2(6):627-46.

Bona, C.A., Casares, S., Brumeanu, T.D. (1998). Towards development of T-cell vaccines. Immunol. Today 19, 126-133.

Louis JM, Nesheiwat I, Chang L, Clore GM, Bewley CA. Covalent trimers of the internal N-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of HIV envelope-mediated cell fusion. J Biol Chem. 2003 May 30;278(22):20278-85.

Ingallinella P, Bianchi E, Finotto M, Cantoni G, Eckert DM, Supekar VM, Bruckmann C, Carfi A, Pessi A. Structural characterization of the fusion-active complex of severe acute respiratory syndrome (SARS) coronavirus. Proc Natl Acad Sci U S A. 2004 Jun 8;101(23):8709-14.

Skehel JJ, Wiley DC. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem. 2000; 69:531-69.