Intranasal vaccine for use against disease caused by enterotoxigenic
United States Patent: 7,666,437
Issued: February 23, 2010
Inventors: Lopez Vidal;
Yolanda (Delegacion Coyoacan, MX), Suaste Villanueva; Olga Roxana (Delegacion
Coyoacan, MX), Godinez Moreno; Ricardo (Delegacion Coyoacan, MX),
Arredondo Hernandez; Luis Jose (Delegacion Coyoacan, MX)
Nacional Autonoma de Mexico (Mexico, MX)
Appl. No.: 12/092,946
Filed: October 26, 2006
PCT Filed: October 26, 2006
PCT No.: PCT/MX2006/000116
371(c)(1),(2),(4) Date: August
PCT Pub. No.: WO2007/073137
PCT Pub. Date: June 28,
Training Courses --Pharm/Biotech/etc.
The disclosure relates to an intranasal
peptide vaccine for use against infection caused by enterotoxigenic
Escherichia coli (ETEC), containing a common linear protein epitope known
as CLE which is recognized by sera of patients infected with the bacteria
and which is associated with a mucosal adjuvant comprising cholera toxin
subunit B, known as CTB. The disclosure relates to a peptide containing
twenty amino acids, which is located in the linear sequence of the CFA/I
fimbria of enterotoxigenic Escherichia coli.
Description of the
FIELD OF THE DISCLOSURE
The present disclosure is related to the use of peptides for the purposes
of vaccination, specifically it relates to peptides that induce a immune
response in mucosa and more specifically it is related to a peptide
included in a intranasal vaccine composition to elicit a protective immune
response in mammals against illness caused by enterotoxigenic Escherichia
BACKGROUND OF THE DISCLOSURE
At present it is estimated that over 1,500 million cases of diarrhea
occurred every year worldwide, of which, 3 million ended in death. Of the
total number of cases of diarrhea, 210 million are caused by the bacterium
enterotoxigenic bacterium Escherichia coli, hereinafter designated as ETEC,
of these episodes 380,000 cases ended in death (World Health Organization
(WHO) State of the art of new vaccines Research & Development Initiative
for Vaccine Research; Geneva, April 2003). Although the diarrhea caused by
this microorganism occurs in groups of all ages, the mortality is more
common in children under 5 years old, particularly when this illness
occurs concomitantly with malnutrition, thereby the developing countries
are specially affected. This bacterium also is the principal causal agent
of the so-called traveler's diarrhea.
ETEC infection is acquired orally, principally through contaminated drinks
or food; the bacterium overcomes the acidic conditions of the stomach
until it reaches the small intestine, where, due to its Colonization
Factor Antigens known as CFA's, it adheres to the intestinal mucosa and
liberates its enterotoxins which are principally two, heat-labile
enterotoxin known as LT and heat-stable enterotoxin or ST which are the
factors responsible for the diarrhea (Gaastra W, Svennerholm A M.
Colonization factors of human enterotoxigenic Escherichia coli (ETEC).
Trends in Microbiology. 1996; 4(11): 444-452).
More than 20 different CFA's have been described in the literature, and of
these three have been identified as the more prevalent, and they are known
as CFA/I, CFA/II and CFA/IV that were reported by several authors, since
there was detected between 50 to 75% of ETEC strains isolated from
patients suffering from diarrhea all over the world, including Mexico, the
majority are grouped in the family CFA/I. (Gaastra W, Svennerholm A M.
Colonization factors of human enterotoxigenic Escherichia coli (ETEC).
Trends in Microbiology. 1996; 4(11): 444-452. Lopez-Vidal Y, Calva J J,
Trujillo A, Ponce de Leon A, Ramos A, Svennerholm A-M, Ruiz-Palacios G M.
Enterotoxins and adhesins of enterotoxigenic Escherichia coli are they a
risk factor for acute diarrhea in the community? J Infect Dis 1990;
162:442-447, and Steinsland H, Valentiner-Branth P, Gjessing H K, Aaby P,
Molbak K, Sommerfelt H. Protection from natural infections with
enterotoxigenic Escherichia coli: Longitudinal study. The Lancet. 2003;
362). These types of CFA's induce protective immunity against the
bacterium and they are pointed out as the most important among the
strategies for the development of effective vaccines. (Middlebrook J L,
Dorland R B. Bacterial Toxins: Cellular mechanisms of Action.
Microbiological Reviews. 1984; 48(3): 199-221. McConell M M, Hibberd M L,
Penny M E, Scotland S M, Cheasty T, Rowe B. Surveys of human
enterotoxigenic Escherichia coli from three different geographical areas
for possible colonization factors. Epidemiol. Infect. 1991; 106: 477-484).
Due to the epidemiological importance of ETEC, many of the efforts have
been directed to the prevention of the illness by obtaining an effective
and safe vaccine, (Levine M M, Kaper J B, Black R E, Clements A M. New
Knowledge on Pathogenesis of Bacterial Enteric Infections as Applied to
Vaccine Development. Microbiological Reviews. 1983; 47(4):510-550)
however, to the date these efforts have been unsuccessful and a product
that satisfies these needs has not been placed in the market yet. (World
Health Organization [WHO] State of the art of new vaccines Research &
Development Initiative for Vaccine Research; Geneva, April 2003).
One of the vaccines that is under development and that has passed to the
phase of trials in human healthy volunteers from different geographical
regions, was prepared in the University of Gotemburg in Sweden, and it is
based on subunit B of cholera toxin combined with 5 formalin inactivated
strains of ETEC, which all together express the CFA's of most
epidemiological importance on a global scale. (Quadri F, Ahmed T, Ahmed F,
Sack B, Sack A, Svennerholm A M. Safety and Immunogenicity of an oral,
inactivated enterotoxigenic Escherichia coli plus cholera Toxin B subunit
vaccine in Bangladeshi children 18-36 months of age. Vaccine 2003;
Other strategies for the development of vaccines have focused on the use
of live bacteria, as it is seen in the work realized in the Center for
Development of Vaccines CVD of the University of Maryland, using Shigella
as vector for the expression of colonization factors and enterotoxins from
ETEC. (Barry E M, Altboum Z, Losonsky G, Levine M M. Immune responses
elicited against multiple enterotoxigenic Escherichia coli fimbriae and
mutant LT expressed in attenuated Shigella vaccine strains. Vaccine 2003;
Recently a vaccine has been prepared with a new technology of
administration; by means of a patch for transdermal immunization
containing the surface component of E. coli known a CS6 and the
heat-labile toxin known as LT; which has been already tested in human
volunteers, where an immune response of Th1 and Th2 type characterized by
IgG2a and IgG1 respectively, was determined (Wenneras C, Firdausi Q,
Prodeep K B, Bladley S and Svennerholm A-M. Intestinal Immune Responses in
patients Infected with Enterotoxigenic Escherichia coli and in vaccinees.
Infect. Immun. 1999; 66:3311-3316. Quadri F, Ahmed T, Ahmed F, Sack B,
Sack A, Svennerholm A M. Safety and Immunogenicity of an oral, inactivated
enterotoxigenic Escherichia coli plus cholera Toxin B subunit vaccine in
Bangladeshi children 18-36 months of age. Vaccine. 2003; 21: 2394-2403.
Byrd, W., and F. J. Cassels. 2003. Mucosal immunization of BALB/c mice
using enterotoxigenic Escherichia coli colonization factors CFA/I and CS6
administered with and without a mutant heat-labile enterotoxin. Vaccine
21: 1884-1893. Helander A, Wenneras C, Quadri F, Svennerholm A M. Antibody
Responses in Humans against Coli Surface Antigen 6 of Enterotoxigenic
Escherichia coli. Infection and Immunity. 1998; 66(9):4507-4510). However,
the immune response against the colonization factors was not consistent as
shown by the prevalence of the illness in different geographical regions.
There is other type of developed strategies that including vaccines formed
by colonization factors encapsulated in microspheres, or by means of the
expression of the subunit B of LT in plants of tobacco, potatoes, tomatoes
and bananas, but these strategies are highly expensive and nevertheless
the conferred protection is low.
Another option can be a vaccine prepared with the common linear epitope of
immuno-dominant sequences from CFA's which offered a broader spectrum and
increases the level of protection from the ETEC infection (Lopez-Vidal Y,
Epitopos continuos y comunes presentes en las fimbrias de Escherichia coli
enterotoxigenica (ETEC). Gac. Med. Mex. 1997; 133 (6): 511-525. Dominguez
M, et al. Colonization Factor Antigenic I Peptide as Intranasal Vaccine
Approach against enterotoxigenic Escherichia coli infection in hamsters.
5th National Symposium, Basic Aspects of vaccines, 1999).
Generally we can recount that different types of vaccines have been
evaluated against diarrhea caused by ETEC and although many of these
vaccines have demonstrated protective effectiveness against ETEC
infection, some of them show certain side effects; another important
problem to be considered in the design of vaccines against ETEC, is the
variability in the prevalence of the different colonization factors, which
prompted us to design a vaccine that is effective and safe against the
different ETEC serotypes.
Intranasal administration can be an additional alternative for a vaccine
against ETEC, which would add additional advantages, providing superiority
over other vaccines. In this respect a great number of studies has been
performed that demonstrated that the mucosa-associated lymphoid tissue
known as MALT, is a common system, that is, that the stimulation in a
certain site of the mucosa also known as inductor site elicits a response
at local level, remotely or in the effector site, this represented a great
advantage in vaccine design, with the form of transmucosal administration
being greatly facilitated (Cripps A W, Kyd J M, Foxwell A R, Vaccines and
Mucosal immunization. Vaccine 2001; 19:2513-2515). The use of the
intranasal route as route of administration has as principal advantages
the presence of a highly vascularized greater surface, the elimination of
the use of syringes with the risk that this poses, the decrease of
administered doses compared to the oral route which involves a decrease of
adverse effects, easy administration, application to a great number of
persons in relatively short times and the induction of antibodies and
cells of the immune response (Zuercher A W. Upper Respiratory Tract
Immunity. Viral Immunology 2003; 3; 279-289). There are studies that
demonstrate that the intranasal administration can stimulate the
production of secretory IgA at intestinal level, which provides a great
advantage in the development of vaccines directed to the protection
against intestinal pathogens (Hong-Yin Wu, Russell M W. Induction of
mucosal and systemic immune responses by intranasal immunization using
recombinant cholera toxin B subunit as an adjuvant. Vaccine 1998;
Vaccine strategies to improve immunity at the level of the mucosa include
the use of these alternate routes together with adjuvants. (Colonization
Factor Antigenic I Peptide as Intranasal Vaccine Approach against
enterotoxigenic Escherichia coli infection in hamsters. Dominguez M, et
al. 5th National Symposium, Basic Aspects of vaccines, 1999). Recently,
nasal mucosa has been used as inductive site that has showed to increase
the immune response at level of the mucosa both locally and distally such
as in intestinal mucosa, respiratory mucosa and genital mucosa.
Administering certain antigen to the mucosa associated to lymphoid tissue
is a way of eliciting immune response in sites distal from mucosa. (Byrd,
W, and F. J. Cassels. 2003. Mucosal immunization of BALB/c mice using
enterotoxigenic Escherichia coli colonization factors CFA/I and CS6
administered with and without a mutant heat-labile enterotoxin. Vaccine
21:1884-1893. Van Ginkel F W, Nguyen H H, McGhee J R, Vaccines for Mucosal
Immunity to Combat Emerging Infectious Diseases. Emerging Infectious
Diseases. 2000; 6(2):123131.)
For the above, is still of great relevance to count with an effective
vaccine against ETEC infection that can be applied globally and through a
route of administration that allows a rapid, easy application and that
could minimize the adverse effects that could be generated.
The present disclosure relates to a peptide vaccine that can be
administered intranasally, containing a common linear protein epitope from
different ETEC colonization factors that can be recognized by sera of
patients infected with said bacteria.
For the development of this vaccine, first the immune response that
elicited the linear common epitope known as CLE associated to mucosal
adjuvant, cholera toxin B subunit known as CTB, in a murine model and in a
Syrian golden hamster model was characterized. Starting from the entire
fimbria CFA/I, a 20 amino acids peptide amino acids named as CLE was
identified and studied, said peptide is located in the linear sequence of
enterotoxigenic Escherichia coli CFA/I fimbria from amino acid 33 to amino
acid 52 herein identified as SEQ ID NO: 1, which contains a terminal
cysteine in position 53, included to form protein complexes such as dimers,
trimers and pentamers, with the trimers being predominant at 37.degree.
C., such that it will have stability in its configuration and its ability
to facilitate recognition by class II receptor from major
histocompatibility complex and T lymphocyte receptor could be increased.
This peptide is capable of eliciting immune protective response against
the principal serotypes of ETEC.
Synthesis of CLE was performed by conventional methods. CTB was purchased
from Merieux Laboratory, France.
In order to elucidate the sequence of peptide CLE, the characterization of
common linear epitopes for different fimbrias of ETEC was carried out, by
means of synthesis of continuous octapeptides spanning the entire sequence
of the colonization factor antigen I designated as CFA/I; using the method
of Geysen in which the synthesis of peptides was carried out in
polyethylene sticks such that each of them corresponded to each of the
wells of an ELISA plate. This arrangement enabled the study of recognition
by antibodies against sera from children under five years old who were
afflicted with ETEC infection, sera of adults from endemic and not endemic
zones, hyperimmune sera that were prepared by immunization of rabbits with
ETEC strains carrying different colonization factors and anti-CFA/1
monoclonal sera. The essay was performed similar to an ELISA. Most of the
sera from children belonging to the convalescent stage recognized all the
octapeptides, with the recognition being higher for three of said
octapeptides, same that were recognized by serum from an adult belonging
to an endemic area. Whereas the anti-CFA/1 monoclonal antibody did not
recognized any peptide. The recognition of octapeptides was designated
The identification of common and continuous epitopes in CFA/I by
hyperimmune heterologous sera was very variable and it depended on each of
the sera, and it was found that the colonization factors with higher
similarity and that exhibited crossed antigenic response were CFA/I, the
surface components of coli 1, 3 and 4 designated as CS1, CS3, CS4 and the
putative colonization factor 0166 or PCF0166 and part of the sequence from
common linear epitope CLE was recognized on these elements. CLE was
selected because it was recognized by 100% of the children's sera after
natural infection and by sera of adults from endemic areas for ETEC. From
this finding, the peptide vaccine CLE of the colonization factors with B
subunit B of cholera toxin CTB was developed in our laboratory, whose
administration was the intranasal route due to the already mentioned
Analysis of CLE Structure
SEQ ID NO: 1
Circular Dichroism: A solution of 100 .mu.g/mL of CLE in trifluoroethane
was used for the readings using a quartz cell of with 1 cm of optical
pathway. Measurements were carried out in a JASCO J-715 spectrophotometer.
The obtained spectrum is the average of five readings.
The spectrum of circular dichroism for CLE in PBS at pH 7.4 and room
temperature 20.degree. C., suggests the formation of a secondary
structure. The spectrum indicates that CLE from CFA possesses a
predominant structure of beta sheet that agrees with the prediction of a
secondary structure performed in the Antheprot 5.0 software.
Dynamic light scattering: One milliliter of 1 mg/ml CLE diluted in 10 mm
PBS, pH 7.4 was used to evaluate the dynamic light scattering at different
temperatures of 5, 15, 25, 35 and 37.degree. C. Hydrodynamic diameter of
CLE was determined using the Zed ziser nano series (Mc Malvern, USA). It
was found that CLE has the ability to form trimeric structures at
physiological conditions of pH and temperature.
Having properly characterized CLE and evaluated its ability to elicit an
protective immunological response, we proceed to develop a vaccine
composition integrated with such a relation in its components, which
enables to potentiate the protective effect of CLE, in this disclosure it
is used preferably in a proportion of 8 parts of CLE to one part of a
suitable adjuvant for use in mucosa, in this case the adjuvant CTB was
used in a pharmaceutically acceptable aqueous vehicle for intranasal
application in a pharmacologically effective dose, without the use of this
adjuvant is limitative, simply it appears like selected in this form of
the disclosure. The proportion used in this vaccine composition will vary
according to the used adjuvant.
The following examples are presented to provide a better understanding of
the subject matter disclosed herein and they are not intended to limit the
disclosure to the same.
Demonstration of the Ability of Peptide CLE to Elicit Protective Immune
Response Against ETEC
Peptide CLE was applied to pathogen-free, 6 to 8 weeks old, female BALB/c
mice, which were randomly distributed into groups of five animals in each
group. Peptide CLE, corresponding to SEQ ID NO: 1, was administered in a
pharmacologically effective dose, that for this case was 50 .mu.g/mL in a
volume of 7.5 .mu.L per mouse, intranasally, three times at 0, 7 and 28
days. The control group was provided with phosphate buffered solution PBS
orally. The challenge was carried out one week after the last
immunization, by administering by gastric route an inoculum of
6.times.10.sup.8 CFU of strain H10407 of ETEC.
To evaluate the conferred protection, parameters such as the excretion of
ETEC in feces by means of coproculture and identification by means of
hemagglutination test in the presence of D-mannose were determined,
(Lopez-Vidal Y. Colonization Factor antigens of Enterotoxigenic
Escherichia coli (Monoclonal Antibodies and Methods for Epidemiological
Studies). 1990. Goteborg, Sweden. Doctoral Dissertation) and the
determination of the presence of diarrhea signs was evaluated according to
the severity with the following scale: 0 no diarrhea; 1 loss of the
consistency of the feces but without wet tail; 2 region perianal and tail
wetted and; 3 tail, paws and low abdomen wetted and with inactive
appearance (Arredondo L J, Zaragoza S, Dominguez M, Willms K, Lopez-Vidal
Y, Cravioto A. Desarrollo de un nuevo Modelo Animal Experimental Hamster
Sirio Dorado para el estudio de la infeccion por Escherichia coli
enterotoxigenica (ETEC). Enf Infec Microbiol. 1997; 17(2):43-46).
We also characterized the immune response obtained for both studied groups
for which cells from the spleen, lymphatic cervical ganglions, inguinal
lymphatic ganglions and Peyer patches were obtained from mice on day 7
after the challenge with ETEC H10407. The adjusted cell suspensions
obtained from each organ, were grown in the presence of antigens of CLE.
At the end of the cellular proliferation, the cells were labeled using
antibodies to identify surface markers such as CD3, CD4 and CD8 and for
the identification of intracellular cytokines such as IFN-gamma, IL-4 and
IL-10. Once the cells were labeled, they were analyzed by flow cytometry.
The results showed that in relation to the excretion of ETEC, it was
observed that on day 1, in both groups of animals positive coprocultures
occurred at 100%, In the third day the positive coprocultures were of 75%
for the control group and 50% in the group immunized with the peptide CLE.
The presence of diarrhea in mice after the infection with ETEC was
observed only with a change of consistency of the feces for the
CLE-immunized and control groups, which continued to the fourth day in the
latter group but from the fifth day, the consistency in feces was normal
in all mice. These results can be appreciated in more detail in FIG. 1 (see Original Patent),
which presents as a summary the follow up of mice after infection with
As for the evaluation of the immune response it was found that the
response of T CD4+ and T CD8+ lymphocytes was slightly higher for the
group immunized with CLE compared to the control group and it is necessary
to emphasize that the increase of T CD4+ lymphocytes was about 2-fold in
Peyer patches with regard to control group as it can be seen in FIG. 2 (see Original Patent).
As for cytokines response, a predominant production of IL-4 and IL-10 was
observed in the group immunized with CLE, an increase being observed in
the production of IL-4 of about 2.6 times in lymphatic cervical ganglions
in the group immunized with CLE relative to the control and of 0.8 times
in the production of IL-10 in the same organ, which is illustrated in FIG.
3 (see Original Patent).
Claim 1 of 13 Claims
1. The isolated peptide common linear
epitope (CLE) consisting of the amino acid sequence set forth in SEQ ID
NO: 1 containing a terminal cysteine, wherein when administered by
intranasal route to mammals, it generates an immune response in Peyer
patches, inguinal, cervical lymphatic ganglions and in the spleen that
confers protection from illness caused by enterotoxigenic Escherichia Coli
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full