WO2017005654A1 - Concomitant dengue, diphtheria, tetanus, whooping cough (pertussis), polio, and haemophilus influenzae type b vaccination. - Google Patents

Abstract

The present invention relates to a combined DTaP-IPV/Hib vaccine for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein said method comprises concomitantly administering said DTaP-IPV/Hib vaccine to a human subject together with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4.

Description

TITLE OF THE INVENTION

Concomitant dengue, diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b vaccination. FIELD OF THE INVENTION

This invention relates to the field of multivalent vaccines.

BACKGROUND OF THE INVENTION

Dengue is the second most important infectious tropical disease after malaria with approximately one-half of the world's population living in areas where there is a risk of epidemic transmission. There are estimated to be 50-100 million cases of dengue fever every year resulting in 500,000 patients being hospitalized for hemorrhagic dengue fever and resulting in approximately 25,000 deaths. Dengue fever virus infections are endemic in more than 100 tropical countries and hemorrhagic dengue fever has been documented in 60 of these countries (Gubler, 2002, TRENDS in Microbiology, 10: 100-103; Monath, 1994, Proc. Natl. Acad. Sci., 91 : 2395- 2400).

Dengue fever is caused by four viruses of the flavivirus genus which are of similar serological type but differ antigenically (Gubler et al, 1988, in: Epidemiology of arthropod-borne viral disease. Monath TPM, editor, Boca Raton (FL): CRC Press: 223-60; Kautner et al, 1997, J. of Pediatrics, 131 : 516-524; Rigau-Perez et al, 1998, Lancet, 352: 971-977; Vaughn et al, 1997, J. Infect. Dis., 176: 322-30). "Dengue fever viruses" or "dengue viruses" are positive single- strand RNA viruses belonging to the Flavivirus genus of the family of flaviviridae. The genomic sequence and organization of the dengue viral genome is well characterized in the art, see, e,g, Sughrue et al (1997) J. General Virology 78(8): 1861-1866. Dengue virus particles are composed of three structural proteins: a genome associated capsid protein (nucleocapsid), a membrane associated protein (M) that is derived during virus maturation by internal cleavage from a glycosylated precursor protein (prM) and a membrane anchored hemagglutinating envelope protein (E). 180 copies each of the E and mature M proteins form a glycoprotein shell around the nucleodapsid. It is believed that E is the major antigenic determinant for serotype specificity, Markoff, J. (1989) J Virol. 63(8):3345- 3352. However, the product of the prM gene, the glycosylated M protein and fragments thereof also possesses antigenic capacity capable of eliciting a specific immune response, Vasquez, et al. (2002) Vaccine 20: 1823-1830. The dengue virus genome comprises a 5' type I end but lacks a 3' poly-A tail. The organization of the genome is as follows: a 5' non-coding region (NCR), a region encoding the structural proteins (capsid (C), pre-membrane/membrane (prM/M), envelope (E)) and a region encoding non-structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and a 3^* NCR. Typical of fiaviviruses, the dengue viral genome encodes an uninterrupted coding region which is translated into a single polyprotein which is post-translationally processed.

Infection with one serotype of dengue may be asymptomatic or may produce a spectrum of clinical disease from non-specific viral syndrome to severe fatal hemorrhagic disease. Dengue fever is characterized by a two-phase fever, headaches, pains in various parts of the body, prostration, eruptions and lymphadenopathy (Kautner et al., 1997, J. of Pediatrics, 131 : 516-524; Rigau-Perez et al., 1998, Lancet, 352: 971-977). The viremic period is of the same length as the febrile period (Vaughn et al., 1997, J. Infect. Dis., 176: 322-30). Cure of dengue fever is complete after 7 to 10 days, but prolonged asthenia is normal. Reduced leukocyte and platelet numbers frequently occur. The effects of dengue virus infection are often more severe in children.

Diphtheria and tetanus are acute infections caused by Corny ebacterium diphtheriae and Clostridium tetani, respectively. The toxins of these bacteria are the major cause of the respective diseases. The vaccines affording protection against these bacteria contain these toxins that are detoxified to lose their infectivity. The toxins are detoxified using chemicals such as formaldehyde or glutaraldehyde [diphtheria toxoid (DT) and Tetanus toxoid (TT)]. CRM 197, a mutant diphtheria toxin, is also used in certain vaccines.

Whooping cough (or pertussis) is caused by Bordetella pertussis. This is a debilitating and serious disease that may even lead to death. The initial vaccines against the disease were based on the whole cells which were inactivated with chemicals such as formaldehyde. Though highly efficacious, such vaccines referred to as 'whole cell pertussis (wP) vaccines', were associated with side effects including fever and local reactions. Accordingly, acellular pertussis (aP) vaccines have been developed.

Poliomyelitis, often called polio or infantile paralysis, is an infectious disease caused by the polio virus. It is a virus which affects the digestive and nervous systems. It causes fever, vomiting and muscle stiffness and can affect the nerves, causing permanent crippling. The disease can paralyse breathing and swallowing muscles, leading to death. Between two and five percent of people with polio die from it and about half of all patients who survive suffer permanent paralysis.

Two different kinds of vaccine are available: a live attenuated (weakened) oral polio vaccine (OPV) developed by Dr. Albert Sabin in 1961 and an inactivated (killed) polio vaccine (IPV) developed in 1955 by Dr. Jonas Salk. IPV, comprising the Salk strains, is given as an injection. The Salk strains are Mahoney type 1, MEF Type 2 and Saukett type 3.

Haemophilus influenzae is a Gram-negative coccobacillus that is a normal part of the upper respiratory tract flora. Haemophilus influenzae type b (Hib) is a major cause of invasive bloodborne infections in young children and a major cause of meningitis in the first 2 years of life. These conditions can develop quickly and if left untreated, they can rapidly cause death. Immunization against Haemophilus influenzae is based on a polysaccharide [polyribose ribitol phosphate (PRP)]. This polysaccharide may be conjugated to a carrier protein, e.g. tetanus protein.

The safest strategy for the above-mentioned diseases prevention remains vaccination. In this regard, some combined DTaP-IPV/Hib vaccines against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b have been developed. In particular, Sanofi Pasteur has developed PENTAXIM^®, which contains diphtheria (D) and tetanus (T) proteins, acellular pertussis (aP) antigens, inactivated poliovirus (IPV) vaccine and Haemophilus influenzae type-b (Hib) capsular polyribosyl-ribitol-phosphate conjugated to tetanus toxoid (PRP-T).

The dengue vaccine candidate currently at the most advanced clinical development stage is a live attenuated tetravalent dengue vaccine developed by Sanofi Pasteur (chimeric yellow fever dengue-tetravalent dengue vaccine or CYD-TDV). The safety and efficacy of this CYD- TDV in preventing dengue disease, based on a vaccination schedule of three doses given 6 months apart, has been demonstrated during the active phases of two Phase III clinical trials in Asia and Latin America (Capeding et al, The Lancet, 384(9951), 1358-1365 (2014) and Villar et al, NEJM, 372(2), 113-123).

Although the availability of multiple pediatric vaccines has alleviated the threat of multiple diseases in the pediatric population, the recommended administration regimen of these vaccines has created an increasingly complex and crowded schedule of vaccinations. The addition of a dengue vaccine to the already crowded childhood vaccination schedule raises issues of compliance with the recommended pediatric vaccination schedule, particularly in those areas of the world where regular healthcare is difficult to obtain. Unfortunately, these same areas are where the threat of dengue fever is particularly acute. One option is to combine multiple vaccines by concomitant administration to enhance compliance with the recommended vaccination schedule. However, the administration of multiple vaccines at a single time also creates issues for effective vaccination. Whenever a multivalent vaccine is administered (or multiple vaccines are concomitantly administered), each individual antigen of the combination induces an immunological response and this can lead to immunological interference. This effect is especially magnified in the case of live attenuated vaccines, since such vaccines must replicate in the host and therefore interference can also occur at the level of viral replication. In fact, when associating live virus vaccines, studies recommend that there is an interval of 30 days between the different vaccines in order to limit any possible interference (Advisory Committee on Immunization Practices (ACIP) - Center for Diseases Control (CDC). General Recommendations on immunization; MMWR Morb Mortal wkly Rep, 1994, Vol. 43(RR-1): 1-38). According to this ACIP-CDC report, the immune response to one live- virus vaccine may be impaired if administered within 30 days of another live- virus vaccine. It can therefore be seen that it is possible to inhibit the immune system's ability to adequately respond to all of the antigens that are concomitantly administered and thus not provide a suitable immune response to one or more of the antigens.

Regarding tetravalent dengue vaccines (which contain a vaccinal strain of each of the four serotypes of dengue), interference may occur between the different strains comprised within the vaccine. For example, association of DENV-2 and DENV-4 in a tetravalent vaccine demonstrated lower immunogenicity as compared with the equivalent monovalent formulations, as was evidenced by geometric mean titers (GMT) (Anderson et ah, 2011, Journal of Infectious Diseases, Vol. 204 : 442-450). Thus, even when concomitantly administering viral vectors of the same disease type, but from distinct virus serotypes, interference leading to lower immunogenicity may occur.

In the context of the present invention, the above-mentioned risk of interference is potentially further exacerbated in view of the fact that the DTaP-IPV/Hib vaccine is designed to induce a protective immune response against five different diseases and the live attenuated tetravalent dengue vaccine contains antigens against the four serotypes of dengue. Beyond the interference effect that is discussed above, increased adverse effects may also be observed when performing concomitant vaccination. Illustratively, Fisker et al. (2014, Vaccine, Vol. 32(5) : 598-605) reported that co-administration of inactivated diphtheria-tetanus pertussis (DTP) vaccine and live attenuated measles vaccines (MV) is associated with increased mortality, as compared with individuals receiving MV only. These authors showed that the same increased mortality was observed when the pentavalent (DTP-H. Influenza type 5-Hepatitis B) vaccine was co-administered with MV and a live attenuated yellow fever vaccine.

In concomitant vaccination, even if the vaccines are administered to sites that are anatomically separate, there is still the potential for interference among the different vaccine agents. The immune system may be overstimulated or inhibited and as a result, may not adequately or optimally respond to the vaccination.

Thus, it flows from the teachings of the prior art that there is high uncertainty of the chance of inducing safe and effective immune protection simultaneously against a plurality of pathogenic viruses and/or bacteria by concomitant vaccination.

SUMMARY OF THE INVENTION

The present invention relates to a combined DTaP-IPV/Hib vaccine for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein said method comprises concomitantly administering said DTaP-IPV/Hib vaccine to a human subject together with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4.

This invention also relates to a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein said method comprises concomitantly administering said dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine.

In particular embodiment, the said methods comprise administration of the dengue vaccine via a three-dose vaccination schedule. In some embodiments, the said methods comprise administration of the dengue vaccine via a three-dose vaccination schedule, the second dose of said dengue vaccine being concomitantly administered with the DTaP-IPV/Hib vaccine.

This invention also relates to a vaccine composition comprising a mixture of a combined DTaP-IPV/Hib vaccine and of a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each serotypes 1 to 4.

In some embodiments, the dengue vaccine comprises a recombinant chimeric virus comprising a yellow fever genomic backbone wherein the preM-E region is the preM-E region of dengue virus.

DESCRIPTION OF THE FIGURES

Figure 1 is a flow chart that depicts the participant vaccination disposition. Figure 2: Geometric mean titers (GMTs) and 95% CIs for each dengue serotypes 1 to 4, 28 days following the second and third CYD-TDV dose according allocated group 1 or group 2 displayed in the figure 1 (i.e. figures 2A and 2B respectively).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have shown that, unexpectedly, the administration of a combined DTaP-IPV/Hib vaccine concomitantly with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 provides protective immunity against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b. Further, the inventors have shown that the concomitant administration of a combined DTaP- IPV/Hib vaccine with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 does not affect the immunogenicity and the safety of the combined DTaP-IPV/Hib vaccine and the dengue vaccine.

It has also been shown by the inventors that the concomitant administration of a combined DTaP-IPV/Hib vaccine with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 results in a protective antibody response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, thus fulfilling the prospective statistical criteria of non-inferiority along with no clinically relevant impact on the safety profile of the combined DTaP-IPV/Hib vaccine in human, especially in small children.

Further, it has been shown herein that the concomitant administration of a combined DTaP-IPV/Hib vaccine with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 results in a neutralising antibody response against dengue, thus fulfilling the prospective statistical criteria of non-inferiority along with no clinically relevant impact on the safety profile of the dengue vaccine in human, especially in small children. In other words, it has surprisingly been shown that, after completion of the dengue immunization regimen, the neutralizing antibody response against dengue following concomitant administration with a combined DTaP-IPV/Hib vaccine is not inferior to the neutralizing antibody response against dengue after sequential administration with a combined DTaP-IPV/Hib vaccine.

Further, it is shown herein that the concomitant administration of a combined DTaP- IPV/Hib vaccine with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 results in a good antibody response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, with no clinically relevant impact on the immunogenicity or safety profile of the combined DTaP-IPV/Hib vaccine. Accordingly, both the DTaP-IPV/Hib and dengue vaccines may therefore be administered to children at the same visit, offering benefits to public health whilst minimizing healthcare resources.

This invention relates to:

- a combined DTaP-IPV/Hib vaccine for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein said method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine to a human subject together with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4,

- a combined DTaP-IPV/Hib vaccine for use in a method for inducing a protective immune response against dengue, wherein said method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine to a human subject together with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4,

- a combined DTaP-IPV/Hib vaccine for use in a method for inducing a neutralising antibody response against dengue, wherein said method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine to a human subject together with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4,

- a combined DTaP-IPV/Hib vaccine for use in a method for inducing a neutralising immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and the four serotypes of dengue, wherein said method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine to a human subject together with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 and wherein the neutralizing immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and the four serotypes of dengue, following completion of the dengue and DTaP-IPV/Hib vaccine administration regimens, is non- inferior to the neutralizing immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and the four serotypes of dengue induced by sequential administration of said combined DTaP-IPV/Hib vaccine and said tetravalent dengue vaccine. Preferably, said dengue vaccine administration regimen is a three dose administration regimen, wherein the combined DTaP-IPV/Hib vaccine is concomitantly administered with the second dose of the dengue vaccine.

- a combined DTaP-IPV/Hib vaccine for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b and a neutralizing immune response against the four serotypes of dengue, wherein said method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine to a human subject together with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 and wherein the neutralizing immune response against the four serotypes of dengue, following completion of the dengue vaccine administration regimen, is non-inferior to the neutralizing immune response against the four serotypes of dengue induced by sequential administration of said combined DTaP-IPV/Hib vaccine and said tetravalent dengue vaccine. Preferably, said dengue vaccine administration regimen is a three dose administration regimen. In particular, said dengue vaccine administration regimen is a three dose administration regimen wherein the combined DTaP-IPV/Hib vaccine is concomitantly administered with the second dose of the dengue vaccine.

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein said method comprises concomitantly administering said dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine,

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, for use in a method for inducing a neutralizing immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein said method comprises concomitantly administering said dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine,

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, for use in a method for inducing a protective immune response against dengue, wherein said method comprises concomitantly administering said dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine,

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, for use in a method for inducing a neutralising antibody response against dengue, wherein said method comprises concomitantly administering said dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine,

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, for use in a method for inducing a neutralising immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and the four serotypes of dengue, wherein said method comprises concomitantly administering said dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine,

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b and a neutralizing immune response against the four serotypes of dengue, wherein said method comprises concomitantly administering said tetravalent dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine and wherein the neutralizing immune response against the four serotypes of dengue, following completion of the dengue vaccine administration regimen, is non-inferior to the neutralizing immune response against the four serotypes of dengue induced by sequential administration of said combined DTaP-IPV/Hib vaccine and said tetravalent dengue vaccine. Preferably, said dengue vaccine administration regimen is a three dose administration regimen. In particular, said dengue vaccine administration regimen is a three dose administration regimen wherein the combined DTaP-IPV/Hib vaccine is concomitantly administered with the second dose of the dengue vaccine.

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 for use in a method for inducing a neutralising immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and the four serotypes of dengue, wherein said method comprises concomitantly administering said tetravalent dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 and wherein the neutralizing immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and the four serotypes of dengue, following completion of the dengue and DTaP-IPV/Hib vaccine administration regimens, is non-inferior to the neutralizing immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and the four serotypes of dengue induced by sequential administration of said combined DTaP-IPV/Hib vaccine and said tetravalent dengue vaccine. Preferably, said dengue vaccine administration regimen is a three dose administration regimen. In particular, said dengue vaccine administration regimen is a three dose administration regimen wherein the combined DTaP-IPV/Hib vaccine is concomitantly administered with the second dose of the dengue vaccine.

- the use of antigens for the manufacture of a combined DTaP-IPV/Hib vaccine for protecting a human individual against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein the said combined DTaP-IPV/Hib vaccine is intended for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, which method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine and a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4,

- the use of antigens for the manufacture of a combined DTaP-IPV/Hib vaccine for protecting a human individual against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein the said combined DTaP-IPV/Hib vaccine is intended for use in a method for inducing a protective immune response against dengue, which method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine and a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4,

- the use of antigens for the manufacture of a combined DTaP-IPV/Hib vaccine for protecting a human individual against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein the said combined DTaP-IPV/Hib vaccine is intended for use in a method for inducing a neutralising antibody response against dengue, which method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine and a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4,

- the use of antigens for the manufacture of a combined DTaP-IPV/Hib vaccine for protecting a human individual against diphtheria, tetanus, whooping cough (pertussis), polio, and

Haemophilus influenzae type b, wherein the said combined DTaP-IPV/Hib vaccine is intended for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and also dengue, which method comprises concomitantly administering said combined DTaP-IPV/Hib vaccine and a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4,

- the use of a live attenuated dengue virus of each of serotypes 1 to 4 for the manufacture of a tetravalent dengue vaccine for protecting a human individual against all four serotypes of the dengue virus, wherein the said dengue vaccine is intended for use in a method for inducing a protective immune response against dengue, which method comprises concomitantly administering a combined DTaP-IPV/Hib vaccine and said dengue vaccine,

- the use of a live attenuated dengue virus of each of serotypes 1 to 4 for the manufacture of a tetravalent dengue vaccine for protecting a human individual against all four serotypes of the dengue virus, wherein the said dengue vaccine is intended for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, which method comprises concomitantly administering a combined DTaP-IPV/Hib vaccine and said dengue vaccine, and

- the use of a live attenuated dengue virus of each of serotypes 1 to 4 for the manufacture of a tetravalent dengue vaccine for protecting a human individual against all four serotypes of the dengue virus, wherein the said dengue vaccine is intended for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, Haemophilus influenzae type b and also dengue, which method comprises concomitantly administering a combined DTaP-IPV/Hib vaccine and said dengue vaccine.

- the use of a live attenuated dengue virus of each of serotypes 1 to 4 for the manufacture of a tetravalent dengue vaccine for inducing in a human individual a neutralizing antibody response against all four serotypes of the dengue virus, wherein the said dengue vaccine is intended for use in a method for inducing a neutralising immune response against dengue, which method comprises concomitantly administering a combined DTaP-IPV/Hib vaccine and said dengue vaccine.

This invention also concerns a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, which comprises concomitantly administering to a human individual a combined DTaP- IPV/Hib vaccine and a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4.

This invention also pertains to a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio and Haemophilus influenzae type b, which comprises:

(i) administering a first dose of a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, and

(ii) concomitantly administering to a human individual a combined DTaP-IPV/Hib vaccine and a second dose of said dengue vaccine, in a time period ranging from 1 month to

9 months following the administration of the first dose of a dengue vaccine.

This invention further relates to a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, which comprises:

(i) administering a first dose of a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4,

(ii) concomitantly administering to a human individual a combined DTaP-IPV/Hib vaccine and a second dose of said dengue vaccine in a time period ranging from 1 month to 9 months following the administration of the first dose of a dengue vaccine, and (iii) administering a third dose of said dengue vaccine which comprises a live attenuated dengue virus in a time period ranging from 1 month to 9 months following the administration of the second dose of said dengue vaccine.

This invention further pertains to:

- a kit comprising a combined DTaP-IPV/Hib vaccine together with instructions for concomitantly administering to a human individual said combined DTaP-IPV/Hib vaccine and a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, and

- a kit comprising a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 together with instructions for concomitantly administering to a human individual said dengue vaccine and a combined DTaP-IPV/Hib vaccine.

By "concomitant administration" or "concomitantly administering" is meant the action of administering at least two products substantially at the same time - that is within 3 days or less and most preferably on the same day, preferably within an interval of a few minutes. If necessary, the at least two products may also be administered some hours apart. For example, the compositions may be administered to an individual within 3 days, 2 days, 24 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes of each other or simultaneously. Advantageously, the at least two products are administered at anatomically separate body sites. In the context of the present invention, two anatomical sites are separate if they are drained by different lymph nodes. For example, the right arm and the left arm are considered to be separate sites. The following separate sites may also be mentioned by way of non-limiting examples: right arm/right thigh; left arm/left thigh; left arm/right thigh. The at least two products may alternatively be mixed together before administration and the resulting mixture is administered at the selected body site, e.g. in the left arm or in the right arm. As a further alternative, the at least two products may be concomitantly administered to the selected body site using a double-barreled syringe. By "sequential administration" or "sequentially administering" is meant the action of administering at least two products not at the same time - that is more than three days separate the administration of the at least two products. For example, two products may be sequentially administered to an individual at least 4 days, 10 days, 15 days, 20 days, 28 days or 30 days apart. Sequentially administered products need not be administered to anatomically separate body sites, although they may be.

Hereinafter, the term "dose" refers to a volume of a vaccine or vaccine composition comprising an "immunoeffective amount" of the antigenic material(s) (i.e. vaccinal virus). An "immunoeffective amount" is an amount of the antigenic material(s) that is sufficient to induce a homologous neutralizing antibody response after the completion of the immunization regime, which is administered to a single individual at a point of time, and especially which is administered to a single human individual.

A dose, composition or vaccine is termed "monovalent" when in addition to a pharmaceutically acceptable excipient, it contains an antigen(s) derived from a single strain or serotype of a microorganism, which is designed to elicit a neutralizing antibody response against that particular strain or serotype of microorganism. A dose, composition or vaccine is termed "multivalent" when it contains antigens from multiple strains or serotypes of a microorganism or antigens from multiple microorganisms. A multivalent dose, composition or vaccine is designed to elicit neutralizing antibodies against multiple strains or serotypes of a microorganism or neutralizing antibodies against different organisms. The nomenclature used is consistent with conventional nomenclature. For example, a dose, composition or vaccine is considered bivalent, trivalent or tetravalent when it contains antigens designed to elicit neutralizing antibodies against two, three or four serotypes of a microorganism or two, three or four different microorganisms respectively. Multivalent compositions may be prepared by simple mixing of monovalent compositions. As used herein, a "tetravalent dengue composition" or "tetravalent dengue vaccine" comprises antigens which induce neutralizing antibodies against all four serotypes 1 to 4 of dengue.

In the context of the present invention, the term "vaccinal dengue composition" refers to a composition comprising vaccinal dengue viruses.

In the context of the present invention, "vaccinal dengue virus" refers to a dengue virus which is capable of inducing neutralizing antibodies against one or more serotypes of dengue virus by the administration of such vaccinal dengue virus to an immunocompetent mammal, e.g. a human. Examples of vaccinal dengue viruses useful in a tetravalent dengue vaccine of the invention include live attenuated dengue viruses. A particular example of a live attenuated dengue virus is a live attenuated chimeric dengue virus. A "live attenuated virus" is a virus which replicates in a permissive host cell but the replicative efficiency of which is reduced relative to the wild-type virus in the same cell type. Attenuated viruses can replicate in a host, but do not induce a disease state associated with the wild-type virus in said host. Examples of live attenuated viruses are known in the art. A live attenuated virus may be prepared, for example, from a wild-type virus by recombinant DNA technology, site directed mutagenesis, genetic manipulation, serial passage, chemical treatment, chemical mutagenesis or electromagnetic radiation. An live attenuated virus useful in the present invention may generate side effects of moderate intensity (i.e. medium to slight, or none) in the majority of vaccinated subjects, while retaining its ability to induce neutralizing antibodies in a mammal, especially in a human subject.

Although attenuated viruses replicate to a lesser degree than wild-type viruses in typical host cells, such attenuated viruses may be produced efficiently in cells which are able to complement functions disrupted in the attenuated virus ("producer cells"). Producer cells may be naturally occurring variants of permissive host cells or may be generated by other means such recombinant DNA technology. In preparing engineered producer cells using recombinant DNA technology, the cell is modified by the insertion of exogenous nucleic acids which complement the functions which are disrupted in the attenuated virus. Such exogenous nucleic acids may be incorporated into the genome of the cell or may be maintained extra-chromosomally.

A live attenuated dengue virus may be derived from dengue virus serotype 1, 2, 3, or 4. In one embodiment, the live attenuated dengue virus is a live attenuated dengue virus that possesses a replicative efficiency in a permissive cell type is at least one order of magnitude less than the wild type virus in the same cell type. In other embodiments, the live attenuated dengue virus is attenuated for replication to a degree of at least two orders of magnitude, three orders of magnitude, four orders of magnitude, five orders of magnitude, six orders of magnitude, seven orders of magnitude or more relative to the wild type virus in the same cell type.

In one embodiment, the vaccinal dengue virus is a live attenuated dengue virus the growth of which at 37°C or 39°C in Huh-7, VERO and/or C6/36 liver cells results in a maximum titer which is at least 10 times less than maximum titer obtained with the wild parent strain under the same culture conditions and as measured using a given method for determining titer. Examples of live attenuated dengue viruses useful in the practice of the present invention include the VDV1 strain, the VDV2 strain, and the strains described for example in applications WO 2002/66621, WO 00/57904, WO 00/57908, WO 00/57909, WO 00/57910, WO 2002/0950075 and WO 2002/102828.

"VDV" or "Vero dengue vaccine" designates an attenuated dengue virus capable of replication in Vero cells and capable of inducing a specific humoral response, including the induction of neutralizing antibodies, in a mammal. VDVl is a virus derived from a live attenuated dengue virus of serotype 1 known as 16007/PDK13, also called LAV1. LAV1 was derived from the wild-type DEN-1 (dengue virus serotype 1) 16007 strain by submitting the wild type strain to 13 passages through primary dog kidney (PDK) cells. LAV1 has been described in EP1 159968 and has been filed with the National Microorganisms Cultures Collection (CNCM, Institut Pasteur, Paris, France) under number 1-2480. VDVl was derived from LAV1 by subsequent adaptation to Vero cells; in this regard, the RNA from LAV1 was extracted and purified before being transfected into Vero cells. The VDVl strain has subsequently been obtained by plate purification and amplification in Vero cells. The VDVl strain has 3 additional mutations in comparison with LAV1. The complete nucleotide sequence of the VDVl strain, as well as a process for preparing and characterizing the VDVl strain have been described in international patent publication WO 2006/134433.

"VDV2" is a strain derived from a live attenuated dengue virus of serotype 2 known as 16681/PDK53, also called LAV2. LAV2 was derived from the wild-type DEN-2 (dengue virus serotype 2) 16681 strain by submitting the wild type strain to 53 passes through PDK cells. LAV2 has been described in EP1 159968 and has been filed with the National Microorganisms Cultures Collection (CNCM, Institut Pasteur, Paris, France) under number 1-2481. VDV2 was derived from LAV2 by subsequent adaptation to Vero cells; in this regard, the RNA from LAV2 was extracted and purified before being transfected in Vero cells. The VDV2 strain was subsequently obtained by plate purification and amplification in Vero cells. The VDV2 strain has 10 additional mutations in comparison with the LAV2 strain, including 3 silent mutations and 1 mutation in a non-coding region. The complete nucleotide sequence of the VDV2 strain, as well as a process for preparing and characterizing the VDV2 strain have been described in the international patent publication WO 2006/134443.

The VDV 1 and 2 strains are prepared by amplification in Vero cells. The viruses produced are harvested and clarified from cell debris by filtration. The DNA is digested by treatment with enzymes. Impurities are eliminated by ultrafiltration. Infectious titers may be increased by a concentration method. After adding a stabilizer, the strains are stored in lyophilized or frozen form before use and then reconstituted when needed.

In the context of the invention, "chimeric dengue virus" or "dengue chimera" means a recipient flavivirus in which the genetic backbone has been modified by exchanging the sequence of at least the envelope (E) protein of the recipient flavivirus by the corresponding sequence of a dengue virus. Alternatively, and more preferably, the genetic backbone of the recipient flavivirus is modified by exchanging the nucleic acid sequences encoding both the pre-membrane (prM) and E proteins of the recipient flavivirus by the corresponding sequences of a dengue virus. Typically, the recipient flavivirus may be attenuated. The recipient flavivirus may be a yellow fever (YF) virus, in which case, the chimera is referred to herein as a "chimeric YF/dengue virus". Preferably, the YF backbone of a chimeric YF/dengue virus according to the present invention is from an attenuated YF virus. The recipient flavivirus may also be a dengue virus and in that case, the chimeric dengue virus is referred to herein as a "chimeric dengue/dengue virus", the dengue virus serotype characteristic of the E or the prM and E proteins being identical or different from the recipient dengue virus serotype characteristic of the genetic backbone. When the recipient flavivirus is a dengue virus, said dengue virus is preferably attenuated. When the serotypes of the recipient and donor dengue viruses are identical, the recipient dengue virus and the donor dengue virus from which the prM and E protein encoding sequences originate are two different virus strains of the same serotype. For use in the present invention, chimeric dengue viruses are typically chimeric YF/dengue viruses. Examples of chimeric viruses useful in the practice of the present invention include the dengue/YF chimeric viruses described in patent application WO 98/37911 and the dengue/dengue fever chimeras such as those described in patent applications WO 96/40933 and WO 01/60847.

In one embodiment, the chimeric YF/dengue virus comprises the genomic backbone of the attenuated yellow fever virus strain YF17D (Theiler M. and Smith H.H. (1937) J. Exp. Med., 65, p 767-786) (viruses YF17D/DEN-1, YF17D/DEN-2, YF17D/DEN-3, YF 1 7D/DEN-4). Examples of YF17D strains which may be used include YF17D204 (YF-VAX®, Sanofi-Pasteur, Swiftwater, PA, USA; STAMARIL®, Sanofi-Pasteur, Marcy TEtoile, France; ARILVAX™, Chiron, Speke, Liverpool, UK; FLAVIMUN®, Berna Biotech, Bern, Switzerland; YF17D-204 France (X15067, X15062); YF17D-204,234 US (Rice et al, 1985, Science, 229: 726-733), or again the related strains YF17DD (Genbank access number U17066), YF17D-213 (Genbank access number U17067) and the strains YF17DD described by Galler et al. (1998, Vaccines, 16(9/10): 1024-1028). Any other attenuated yellow fever virus strain which may be used in man may be used to construct chimeras in the context of this invention.

One example of a chimeric YF/dengue virus suitable for use in the practice of the present invention is the "Chimerivax™ dengue" or "CYD", a chimeric yellow fever (YF) virus which comprises the genomic backbone of an attenuated YF virus in which the sequences coding for the pre-membrane (prM) and envelope (E) proteins have been replaced by nucleic acid sequences encoding the corresponding structural proteins of a dengue virus. Construction of chimeric Chimerivax virus may be achieved in substantial accordance with the teaching of Chambers, et al. (1999) J Virology 73(4):3095-3101. A chimeric dengue virus containing the prM and E sequences of a serotype 1 dengue fever strain (DEN-1) is referred to as "CYD-1 or CYD DEN1 ". A chimeric YF containing the prM and E sequences of a DEN-2 strain is referred as "CYD-2 or CYD DEN2". A chimeric YF virus containing the prM and E sequences of a DEN-3 strain is referred to as "CYD-3 or CYD DEN3". A chimeric dengue virus containing the prM and E sequences of a DEN-4 strain is referred to as "CYD-4 or CYD DEN4". The preparation of these dengue Chimerivax™ viruses have been described in detail in international patent applications WO 98/37911 and WO 03/101397, to which reference may be made for a precise description of the processes for their preparation. The chimeras described in the examples have been generated by using prM and E sequences from strains DEN 1 PU0359 (TYP1 140), DEN2 PU0218, DEN3 PaH881/88 and DEN 4 1228 (TVP 980). Alternatively, other dengue fever virus strains may be used as a source of nucleic acids to facilitate construction of chimeric viruses useful in the practice of the present invention. The sequences SEQ ID NOs 1 to 4 corresponding to the nucleotide sequences of the prM-E regions of the chimeras of serotypes 1 to 4 described in the examples are set out in the Table below. The sequence SEQ ID N°5, corresponding to the nucleotide sequence of the prM-E region of different serotype 2 strain (MD1280), is set out in the Table below under the designation "CYD-2V". Sequences having at least 90% sequence identity to the sequences of SEQ ID NOs 1-5 may be used as a source of nucleic acids to facilitate construction of chimeric viruses useful in the practice of the present invention. Use of CYD-2V in combination with chimeric YF/DEN strains of serotypes 1, 3 and 4 which are respectively based on the prM-E sequences of SEQ ID NOs 1, 3 and 4 results in a tetravalent dengue vaccine that produces a more balanced neutralizing immune response across the four serotypes (as described in WO 2014/016,362).

Alternatively, other dengue fever virus strains may be used as a source of nucleic acids to facilitate construction of chimeric viruses useful in the practice of the present invention.

An example of a live attenuated dengue virus of serotype 1 virus useful in the present invention may for example be the strain VDV1, a Chimerivax™ DEN- lor a YF17D/DEN-1 chimeric virus comprising prM and E genes of the DEN-1 16007/PDK13 strain. An example of a live attenuated dengue virus of serotype 2 useful in the present invention is the strain VDV2, a Chimerivax™ DEN-2 (e.g. based on SEQ ID NO. 2 or SEQ ID NO. 5) or a YF17D/DEN-2 chimeric virus comprising prM and E genes of the DEN-2 16681/PDK53 strain. An example of a live attenuated dengue virus of serotype 3 is a Chimerivax™ DEN-3 or a YF17D/DEN-3 chimeric virus. An example of a live attenuated virus of serotype 4 is a Chimerivax™ DEN-4 or a YF17D/DEN-4 chimeric virus. The skilled artisan may refer to the aforementioned published international patent applications for a detailed description of the strains mentioned, the processes for obtaining them and the construction of these chimeric viruses. The SEQ ID NOs 6 and 7 corresponding to the nucleotide sequences of the above-mentioned VDV1 and VDV2 strains are set out in the Table below.

The detection of serum antibodies to dengue serotypes, diphtheria, tetanus, pertussis, polio and Hib are well known in the scientific literature. Examples of such assays are described in Example 1 below.

The ability of a dengue vaccine composition of the present invention to provoke an immune response in a subject (i.e. induce the production of neutralizing antibodies) can be assessed, for example, by measuring the neutralizing antibody titre raised against the dengue virus serotype(s) comprised within the composition. The neutralizing antibody titre may be measured by the Plaque Reduction Neutralization Test (PRNT50) test as described in Example 1 below. It has been commonly considered that seroconversion occurs when the titre is superior or equal to 10 (1/dil). As PRNT tests may slightly vary from a laboratory to another the LLOQ may also slightly vary. Accordingly, in a general manner, it is considered that seroconversion occurs when the titre is superior or equal to the LLOQ of the test. Neutralising antibody titres were considered in the following references, but the authors did not establish a correlate of protection (Guirakhoo et al, J. Virol. (2004) 78 (9): 4761; Libraty et al, PLoS Medicine (2009) 6 (10); Gunther et al, Vaccine (2011) 29: 3895) and Endy et al, J. Infect. Dis. (2004), 189(6): 990-1000).

A DTaP-IPV/Hib vaccine according to the present invention comprises a mixture of antigens which are able to induce protection against diphtheria, tetanus, whooping cough (pertussis), polio and infections caused by Haemophilus influenzae type b. Preferably, the DTaP- IPV/Hib vaccine comprises a diphtheria toxoid; a tetanus toxoid; an acellular pertussis antigen; an inactivated poliovirus (IPV) and a Haemophilus influenza type b capsular polysaccharide covalently bound to tetanus toxoid.

More preferably, the diphtheria and tetanus toxoids are detoxified.

More preferably, the acellular pertussis antigen is selected from the group consisting of a detoxified pertussis toxoid, a filamentous haemagglutinin or a combination thereof.

More preferably, the acellular pertussis antigen is a combination of a detoxified pertussis toxoid and a filamentous haemagglutinin which have been separately adsorbed onto aluminium hydroxide .

More preferably, the inactivated poliovirus (IPV) may comprise at least one antigen selected from a group comprising the IPV type-1 (Mahoney strain), the IPV type-2 (MEF-1 strain), the IPV type-3 (Saukett strain) or a combination thereof.

More preferably, the Haemophilus influenza type b capsular polysaccharide may be a polyribosylribitol phosphate.

According to a preferred embodiment, a DTaP-IPV/Hib vaccine according to the present invention is a pharmaceutical formulation comprising, and preferably containing, in addition to excipients and aluminum hydroxide adjuvant, at least 30 IU detoxified diphtheria toxoid, at least 40 IU detoxified tetanus toxoid, approximately 25 micrograms of detoxified Bordetella pertussis toxoid and filamentous hemagglutinin antigens, approximately 40 DU of inactivated Type 1 (Mahoney) poliomyelitis virus, approximately 8 DU of inactivated Type 2 (MEF-1) poliomyelitis virus, approximately 32 DU of inactivated Type 3 (Saukett) poliomyelitis virus, and approximately 10 micrograms of the polysaccharide of Haemophilus influenzae type b (polyribosylribitol phosphate) conjugated to detoxified tetanus toxin in a volume of 0.5 ml (such a pharmaceutical formulation is notably described in EP 2 353 609). For use in the present invention, a DTaP-IPV/Hib vaccine may be any DTaP-IPV/Hib commercialized vaccine. Examples include the DTaP-IPV/Hib vaccine commercialized under the names PENTACEL^® and PENTAXIM^® by Sanofi Pasteur or INFANRIX^®-IPV/Hib developed by GlaxoSmithKline AG.

The immunogenicity of the DTaP-IPV/Hib vaccine may be assessed as described in the examples below.

In some embodiments, the present invention relates to a combined vaccine composition comprising a mixture of a combined DTaP-IPV/Hib vaccine and of a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of the 4 dengue serotypes. Various embodiments of a combined DTaP-IPV/Hib vaccine and of a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of the four serotypes of dengue are described elsewhere in the present specification.

In some embodiments, the said combined vaccine composition comprises a mixture of:

- a combined DTaP-IPV/Hib vaccine in an amount sufficient for inducing a protective immune response in a human subject, and

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, in an amount sufficient for a second dose of a dengue vaccine adapted for a three-dose dengue vaccination schedule in a human subject.

In some embodiments, the said combined vaccine composition is manufactured as a ready-to-use vaccine. In some other embodiments, the said combined vaccine is prepared a short time period, e.g. one hour or less, before administration to an individual, for example by simply mixing a dose of a dengue vaccine as described herein with a dose of a combined DTaP-IPV/Hib vaccine as described herein and then administering the resulting vaccine mixture to the said individual, whereby the combined DTaP-IPV/Hib vaccine and the dengue vaccine are subject to concomitant administration. This invention also relates to a combined vaccine comprising a combined DTaP-

IPV/Hib vaccine and a dengue vaccine as described herein for use in a method of inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b (Hib), wherein said method comprises administering the said combined vaccine. As it shall be readily understood form the whole disclosure herein, the said combined vaccine is used only once in a method for inducing a protective immunity of a human individual against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b (Hib), and also dengue. Typically, the combined vaccine composition is used for the second injection, i.e. the step of concomitant administration, in a three dose dengue vaccination schedule, which is preceded by one administration of a dengue vaccine which comprises a live attenuated dengue virus and which is followed by one administration of a dengue vaccine which comprises a live attenuated dengue virus, in a time schedule that is specified elsewhere in the present specification. The exact quantity of a live attenuated dengue virus or a live attenuated chimeric dengue virus of the present invention to be administered may vary according to the age and the weight of the human subject being vaccinated, the frequency of administration as well as the other ingredients in the composition. Generally, the quantity of a live attenuated dengue virus (e.g. VDV1 or VDV2) comprised in a dose of a vaccine composition as used in a method as described herein lies within a range of from about 10³ to about 10⁶ CCID50, for example within a range of from about 5 x 10³ to about 5 x 10⁵, for example about 10⁴ CCID50. The quantity of a live attenuated chimeric dengue virus (such as a chimeric YF/dengue virus or a Chimerivax® (CYD) virus) comprised in a vaccine composition as used in the method of the present invention lies within a range of about 10⁵ CCID50 to about 10⁶ CCID50. The quantity of a live attenuated dengue virus or live attenuated chimeric dengue virus of each of serotypes 1 to 4 comprised in a tetravalent dosage form according to the present invention is preferably equal. The term CCID50 refers to the quantity of virus infecting 50% of the cell culture. The CCID50 assay is a limit dilution assay with statistical titer calculation (Morrison D., et al., J. Infect. Dis. (2010), 201(3): 370-377). The vaccinal compositions of the present invention may also include one or more pharmaceutically acceptable vehicles. The term "vehicle" refers to compounds commonly used in the formulation of pharmaceuticals and vaccines to enhance stability, sterility and deliverability of the active agent. Suitable vehicles and their preparation are described, for example, in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol, Ed., Mack Publishing Co., Easton, PA (1980), and Remington's Pharmaceutical Sciences, 19th Edition, A.R. Gennaro, Ed., Mack Publishing Co., Easton, PA (1995). When the vaccinal composition is formulated as a solution or suspension, the immunologically active agent is provided in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques including sterile filtration via a 0.2 micron pore filter. The resulting aqueous solutions may be packaged for use. Alternatively, the aqueous solutions may be lyophilized, the lyophilized preparation being reconstituted with a sterile aqueous solution prior to administration.

The vaccinal compositions may optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorption monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, nonessential amino acids, L-arginine hydrochlorate, saccharose, D-trehalose dehydrate, sorbitol, tris (hydroxymethyl) aminomethane and/or urea. In addition, the vaccinal composition may optionally comprise pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. A preferred example of a stabilizing solution which may be used in the context of the tetravalent live attenuated dengue vaccine as described herein is disclosed in WO 2010/003670. According to a particular embodiment of a combined DTaP-IPV/Hib vaccine composition, the said vaccinal composition contains (per 0.5 mL dose) the amounts of antigens as described above in combination with aluminium hydroxide (hydrated for adsorption, 0.6 mg Al³⁺) and 15 mg of buffer components comprising disodium hydrogen phosphate, potassium dihydrogen phosphate, essential amino acids, trometamol and saccharose. Unit dosage formulations of the vaccinal compositions of the present invention may be included in a kit of products containing the vaccinal virus(es) in lyophilized form and a solution for reconstitution of the lyophilized product. Recombinant viruses of the present invention may be lyophilized by conventional procedures and reconstituted. Such solutions for reconstitution of the lyophilized vaccinal composition may be aqueous solvents comprising buffers, organic or inorganic salts, and agents to assist in solubilization.

The concomitant administration of the vaccinal compositions of the present invention may be achieved by transcutaneous, subcutaneous, intramuscular or intradermal injection. Preferably a dengue vaccine according to the present invention is administered subcutaneously and a DTaP-IPV/Hib according to the present invention is administered intramuscularly. Preferably the two vaccines are administered at anatomically separate sites as previously described. The vaccinal compositions may be administered using conventional hypodermic syringes or safety syringes such as those commercially available from Becton Dickinson Corporation (Franklin Lakes, NJ, USA) or jet injectors. For intradermal administration, conventional hypodermic syringes may be employed using the Mantoux technique or specialized intradermal delivery devices such as the BD Soluvia™ microinjection system (Becton Dickinson Corporation, Franklin Lakes, NJ, USA), may also be employed.

In one embodiment, the human subject is aged between 12 months and 60 years old, between 2 and 60 years old, between 6 and 60 years old, between 9 and 60 years old, between 12 months and 16 years old, between 2 and 16 years old, between 6 and 16 years old or between 9 and 16 years old. Advantageously, the human subject is at least 12 months of age, at least 2 years of age, at least 5, 6, 7, 9, 12 or 15 years of age. Advantageously, the human subject is at least 9 years of age, e.g. between 9 and 60 years old or between 9 and 45 years old.

Preferably said human subject is dengue immune and/or said human subject resides in a dengue endemic area. As used herein, a "dengue immune" subject refers to a subject who has been infected by a dengue virus or immunized by a dengue vaccine before administration of the vaccine composition of the present invention, i.e. a serum sample taken from said subject will produce a positive result in a dengue ELISA or PRNT50 assay. Dengue endemic areas are well- known to a person of skill in the art and include, according to the present invention, most of the tropics and sub-tropics, for instance any country identified as an endemic country by the WHO. A dengue endemic area may be defined herein as an area in which the population is at least 50% dengue immune or at least 60% dengue immune. More preferably, a dengue endemic area may be defined as an area in which the population is at least 70%> dengue immune, at least 80%> dengue immune or at least 90% dengue immune. An area can also be defined as dengue-endemic based on vector presence, the co-circulation of multiple serotypes in the area and the sustained transmission of the disease indicated through routine surveillance data. For example, a dengue endemic area may be defined as an area in which the population is dengue immune as described above, wherein the dengue vector is present and wherein there is sustained transmission of the disease, optionally with co-circulation of multiple serotypes.

In one embodiment, the invention provides a multi-step dosage regimen. An initial administration of a dengue vaccine composition is performed at a time TO and is followed by the concomitant administration of a second dose of the vaccinal dengue composition and of the combined DTaP-IPV/Hib vaccinal composition at a date approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 months following TO (this second co-administration being administered on a date termed Tl).

In another embodiment, a third administration of the vaccinal dengue composition may be administered at a date approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 months following Tl (this third administration being administered on a date termed T2). Preferred two dose regimens include 0-3 and 0-6 (i.e. a first dose of the dengue vaccinal composition at TO, followed by a second dose of the dengue vaccinal composition concomitantly administered with the combined DTaP-IPV/Hib vaccine at Tl, which is 3 or 6 months after TO). Most preferred is a three dose regimen (i.e. a three-dose vaccination schedule) for the dengue vaccinal composition. Examples of such three dose regimens include 0-6-12 and 0-2-6 (i.e. a first dose of the dengue vaccinal composition at TO, followed by a second dose of the dengue vaccinal composition concomitantly administered with the combined DTaP-IPV/Hib vaccine at Tl, which is 2 or 6 months after TO and a third dose of the dengue vaccinal composition at T2, which is 6 or 4 months after Tl .

In a preferred embodiment of the invention, the dose of the combined DTaP-IPV/Hib vaccine which is concomitantly administered with a dengue vaccine according to the invention is a booster dose of the DTaP-IPV/Hib vaccine, which follows an initial three dose regimen of the DTaP-IPV/Hib vaccine administered in the first year of life, e.g. between 2 and 8 months of age. In such an embodiment, the booster dose of the DTaP-IPV/Hib vaccine is administered during the second year of life (i.e. between the ages of 12 and 24 months). Booster administrations of the vaccinal dengue compositions and/or (further) boosters of the combined DTaP-IPV/Hib vaccine may be administered subsequent to the foregoing dosage regimen to maintain robust immunoprotection in the human. Such booster administrations may occur at time points of approximately 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer after the initial vaccination regimen.

According to another aspect, this invention has as its object a kit.

The kit according to the invention comprises vaccinal compositions as described in relation to the method described herein. The kit according to the invention therefore comprises a box containing various containers holding the compositions or vaccines and advantageously an explanatory brochure including useful information for administration of the said compositions or vaccines. The term container includes conventional sealed vials and prefilled syringes. According to one embodiment, this invention therefore relates to a kit for immunization against dengue serotypes 1, 2, 3, and 4, as well as diseases such as diphtheria, tetanus, acellular pertussis, and infections caused by Haemophilus influenzae and polio viruses, comprising a box containing at least (a) a first container holding a combined DTaP-IPV/Hib vaccine, and (b) a second container holding a dengue vaccine as described herein. The vaccinal compositions which may be used in the kit according to the invention include the vaccinal compositions described herein in relation to the method according to the invention.

If the vaccinal compositions are provided in lyophilized form, the kit will advantageously comprise at least one additional container holding a solution which can be used to reconstitute a lyophilized vaccinal composition suitable for administration by intradermal, transcutaneous, subcutaneous, or intramuscular administration. Pharmaceutically acceptable diluents and carriers may be used for reconstitution.

According to a particular embodiment, the kit according to the invention comprises a tetravalent vaccine comprising 10⁵ to 10⁶ CCID50 of Chimerivax™ DEN-1, 2, 3 and 4. The container in which the pharmaceutical formulation is packaged prior to use can comprise a hermetically sealed container enclosing an amount of the lyophilized formulation or a solution containing the formulation suitable for a pharmaceutically effective dose thereof, or multiples of an effective dose. The pharmaceutical formulation is packaged in a sterile container, and the hermetically sealed container is designed to preserve sterility of the pharmaceutical formulation until use.

Optionally, the container can be associated with administration means and or instruction for use. Examples of administration means may include syringes for parenteral administration or delivery systems to facilitate intradermal administration.

Regarding the pharmaceutical dosage forms, the volume of vaccinal composition administered will depend on the method of administration. In the case of subcutaneous injections, the volume is generally between 0.1 and 1.0 ml, preferably approximately 0.5 ml. In the case of intramuscular injections, the volume is generally between 0.1 and 1.0 ml, preferably approximately 0.5 ml.

This invention is further illustrated by, without in any way being limited to, the examples below.

Examples

Methods

Study design and participants

This was a randomized, observer-blind (for the second dose of CYD-TDV), open- label (for the first and third doses of CYD-TDV), multi-center, Phase III trial conducted in 732 healthy toddlers in Mexico between 18 July 2011 and 4 February 2014. This trial was conducted in accordance with the standards established by the Declaration of Helsinki and the International Conference on Harmonisation (ICH) guidelines for good clinical practice (GCP) as well as with all local and/or national regulations and directives. In addition, the study protocol, including any amendments, was approved by each study site's Institutional Review Board and Independent Ethics Committee. Written informed consent was obtained from all participants' parents/guardians before study entry.

Eligible participants were toddlers aged 9 to 12 months in good health, based on medical history and physical examination. In addition, they had to be born at full term (> 37 weeks) and with a birth weight > 2.5 kg, and had to have documented evidence of completion of the primary vaccination series with DTaP-IPV//Hib vaccine (Pentaxim^®; Sanofi Pasteur S.A., France) with 3 doses received between 2 and 8 months of age. Participants were excluded if they had previous history of pertussis and/or Hib infection or if they had previous vaccinations against flavivirus diseases, measles, mumps, rubella, previous booster vaccination against pneumococcal diseases, diphtheria, tetanus, pertussis, Hib and/or polio. Other exclusion criteria included: known or suspected congenital or acquired immunodeficiency; receipt of immunosuppressive therapy such as anti-cancer chemotherapy or radiation therapy within the preceding six months, or long-term systemic corticosteroid therapy (for more than two consecutive weeks within the previous three months); receipt of blood or blood-derived products in the past three months; history of central nervous system disorder or disease, including seizures; thrombocytopenia, bleeding disorders or receipt of anticoagulants in the three weeks preceding inclusion; known systemic hypersensitivity to any of the components of the vaccines; seropositivity to human immunodeficiency virus or hepatitis C.

Randomization and blinding

Participants were allocated to two subsets; the viremia subset or the dengue immunogenicity subset. Participants could not be allocated into both the dengue immunogenicity subset and the viremia subset due to restrictions in the volumes of blood samples that were permitted. The first 100 participants enrolled in the trial formed the viremia subset, whereas subsequent participants were randomly assigned to the dengue immunogenicity subset (or not). A total of 250 participants were allocated to the dengue immunogenicity subset. At month 6, the participants were randomized in a 1 : 1 ratio into two vaccine groups (Group 1 or Group 2) via an interactive voice response system or interactive web response system. Randomization was performed with permuted block method with stratification by center, age group (participants aged 9 to 10 months versus 11 to 12 months), and inclusion in the dengue immunogenicity subset, using a double randomization procedure.

Vaccination and vaccines

CYD-TDV (Sanofi Pasteur S.A., France) tetravalent live attenuated vaccine comprising a chimeric yellow fever-dengue strain of each of the four serotypes of dengue, wherein for each serotype of dengue, the genomic sequence encoding the prM and E proteins of an attenuated YF strain have been replaced with the equivalent sequences (one of SEQ ID NOs 1- 4) from a dengue virus; Batch numbers: S4316F09; S4316F13; S4316F19; and S4395F04) was presented as a powder and saline solvent (NaCl 0.4%) for reconstitution immediately before use in 0.5 mL volumes containing 5±1 logio cell-culture infectious dose 50% (CCID50) of each of the four live attenuated recombinant CYD-TDV virus serotypes. CYD-TDV was administered subcutaneously in the deltoid region of the upper arm.

DTaP-IPV//Hib vaccine (PENTAXIM®, Sanofi Pasteur, France) was supplied as powder and suspension for injection. Each 0.5 mL dose of reconstituted vaccine contains >30 IU of diphtheria toxoid, >40 IU of tetanus toxoid, 25 μg of pertussis toxoid (PT), 25 μg of filamentous hemagglutinin (FH), 40 DU of inactivated polio virus (IPV) type 1 , 8 DU of IPV type 2, 32 DU of IPV type 3 and 10 μg of polysaccharide of Haemophilus influenzae type b conjugated to the tetanus protein. The DTaP-IPV//Hib vaccine was administered intramuscularly, to the anterolateral aspect of the thigh and the placebo (0.5 ml) was administered subcutaneously to the deltoid region of the upper arm.

All participants received the first dose of CYD-TDV (Sanofi Pasteur) at enrollment (month 0). At 6 months (at 15 to 18 months of age), participants allocated to Group 1 received the booster dose of DTaP-IPV//Hib vaccine (PENTAXIM®, Sanofi Pasteur) concomitantly with the second dose of CYD-TDV using an observer-blind procedure. A placebo injection was administered at month 7 to maintain the blind, and the third dose of CYD-TDV was administered at month 12 using an open- label procedure. Those randomized to Group 2 received the booster dose of DTaP-IPV//Hib vaccine concomitantly with a placebo injection at month 6, with the second dose of CYD-TDV administered at month 7 and the third dose at month 12. The three CYD-TDV injections were administered alternatively in the right and left limbs. All participants also received a measles, mumps, and rubella (MMR) vaccination and pneumococcal conjugated vaccine at month 1 (at 10 to 13 months of age), as recommended in the immunization schedule of Mexico. However, no assessment of safety or immunogenicity was conducted for these two vaccines.

Immunogenicity assessments

All participants provided one pre-vaccination blood sample at enrollment to assess baseline dengue immune status before the first vaccination; one blood sample before DTaP- IPV//Hib vaccination at month 6 for assessment of baseline pertussis antigens (PT and FHA); and one post-vaccination blood sample at month 7 for determination of DTaP-IPV//Hib vaccine immunogenicity. Antibody levels against diphtheria toxoid, tetanus toxoid and pertussis antigens (PT and FHA) were measured by Enzyme-Linked Immunosorbent Assay (ELISA) at Sanofi Pasteur GCI, Swiftwater, USA. Briefly, serum samples were added to wells of four separate ELISA microtiter test plates coated with either diphtheria, tetanus, PT or FHA antigens. Serum samples (test samples, reference standard, and quality control) were incubated in the wells. The lower limit of quantification of the assay (LLOQ) of the assay was 0.007 IU/mL for diphtheria IgG, 0.01 IU/mL for anti-tetanus, and 2 EU/mL for anti-PT and anti-FHA. Anti-polio virus types 1, 2, and 3 titers were measured by neutralization assay by Sanofi Pasteur GCI, Swiftwater (Expanded Program on Immunization and Division of Communicable Diseases. Manual for the virological investigation of poliomyelitis. Geneva: World Health Organization. 1990. Document WHO/EPI/CDS/Polio/90.1 and Albrecht P, J. Bio. Stand. 1983; 11 :91-97). The LLOQ of the anti- poliovirus types 1, 2, and 3 assays was 4 (1/dil). Anti-PRP (Hib) antibodies were measured using a Farr-type radioimmunoassay and compared to a reference standard (Anderson P., J. Immunol. (1978); 120(3):866-70); the LLOQ of the assay was 0.06 μg/mL. Neutralizing antibody responses against each of the four parental dengue virus serotype strains of the CYD-TDV constructs were measured 28 days after administration of the second and third CYD-TDV dose, using the plaque reduction neutralization test (PRNT50, Timiryasova TM, Am. J. Trap. Med. Hyg. 2013 May; 88(5):962-70). Briefly, serial, 2-fold dilutions of serum to be tested (previously heat-inactivated) were mixed with a constant challenge-dose of each dengue virus serotype dengue- 1, -2, -3 or -4 (expressed as plaque- forming units [PFU]/mL). The mixtures were inoculated into wells of a microplate with confluent Vero cell monolayers. After adsorption, cell monolayers were incubated for a few days. The presence of dengue virus infected cells was indicated by formation of plaques. A reduction in virus infectivity due to neutralization by Ab present in serum samples was detected. The reported value (end point neutralization titer) represented the highest dilution of serum at which > 50% of dengue challenge virus (in plaque counts) was neutralized when compared to the mean viral plaque count in the negative control wells which represented the 100% virus load. The end point neutralization titers were presented as continuous values. The lower level of quantitation (LLOQ) of the assay was 10 (1/dilution [dil]).

Dengue vaccine viremia

The occurrence and level of dengue vaccine viremia was measured in the serum of participants in the viremia subset (100 participants), 8 days after the first CYD-TDV dose by quantitative and serotype-specific reverse transcriptase polymerase chain reaction (RT-PCR) (Sanofi Pasteur GCI, Swiftwater, USA; Mantel N, J. Virol. Methods. 2008 Jul; 151(l):40-6). First, quantitation of non-serotype specific vaccine viremia was determined using RT-PCR with primers from the yellow fever core gene sequence. For serum samples positive for yellow fever, four further RT-PCRs were performed with serotype-specific primers from the envelope-non- structural (NS) protein 1 junction gene sequence, to enable identification and quantitation of serotype-specific CYD dengue vaccine viremia. Based on the dengue virus plasmidic standards included in each run, results were expressed as a concentration of logio GEq/mL.

Safety and reactogencity

Participants were kept under observation for 30 minutes after each trial vaccination to assess the occurrence, intensity and relationship to vaccination of any immediate injection site or systematic adverse events (AEs). Parents/guardians were provided with a digital thermometer, flexible ruler and diary cards to record daily temperature and any solicited local injection site reactions (erythema and/or swelling) on the day of vaccination and for the next 7 days. They also recorded any solicited systemic reactions for the 14-day period following vaccination, until resolution. Measurable adverse reactions of erythema, swelling and fever were graded on a three point scale during statistical analysis. In addition to solicited reactions, parents/guardians recorded any other medical events (unsolicited AEs) that occurred during the 28-day period after each vaccination. The intensity of unsolicited AEs were graded using a 3 -point scale (Grade 1 : No interference with activity; Grade 2: Some interference with activity; and Grade 3: Significant; prevents daily activity) and the action taken for each event, if any (e.g. medication, contact healthcare provider, medication prescribed or hospitalization). The investigators assessed the causal relationship of each unsolicited systematic AE and, all serious AEs (SAEs), from inclusion in the study until 6 months after the last vaccination, and assigned them as either related or unrelated to vaccination.

Serious dengue disease (severity assessed according to WHO classification) (World Health Organization, Dengue Guidelines for diagnosis, treatment, prevention and control 2009), occurring at any time during the study, was reported as an SAE. Suspected serious dengue disease was defined as febrile episodes (temperature > 38°C for > 2 days) and with signs of severity requiring hospitalization. In such cases, 2 blood samples were collected for further analysis: an acute sample (0-5 days after fever onset) and a convalescent sample (7-14 days post-acute sample). Viro logically-confirmed dengue was defined as detection of wild-type dengue virus by NS1 antigen ELISA (Platelia™, Bio-Rad Laboratories) and/or amplified genomic sequences through RT-PCR (Callahan JD et al, Journal of Clinical Microbiology. 2001; 39:4119-4124). Probable dengue was defined as the presence of anti-dengue immunoglobulin M (IgM) and/or a 4- fold or greater rise in anti-dengue IgG antibody titers between acute and convalescent samples (excluding cases occurring within 28 days after each vaccination). IgM and IgG were measured by ELISA (ELI1500M and EL1500G; Focus Diagnostics, California, USA).

Statistical Analysis

The primary objective of this study was to test the non-inferiority of the antibody response against diphtheria, tetanus, pertussis, polio and Hib antigens in participants receiving one booster dose of DTaP-IPV//Hib vaccine administered concomitantly with the second dose of CYD-TDV compared to those receiving one booster dose of DTaP-IPV//Hib vaccine concomitantly with placebo. Based on simulations using the Wilson score method (without continuity correction) and, assuming a 15% drop out rate, an alpha=2.5% (one-sided), reference antibody response rates and 90% power, then a total of 732 participants (366 per group) were needed to be enrolled (310 per group evaluable). With 366 participants per group, the probability to detect any common AEs with an incidence of >0.82% was 95%. Reference antibody response rates were obtained from post-booster results from comparative clinical trials of penta- and hexavalent vaccines including at least 100 subjects per group, and were as follows: tetanus > 0.1 IU/mL, 99%; diphtheria > 0.1 IU/mL, 99%; IPV1 >8 (1/dil), 99%; IPV2 >8 (1/dil), 99%; IPV3 >8 (1/dil), 99%; PRP >1 g/mL, 98%; PT >4-fold increase in post-vaccination titres, 95%; FHA >4-fold increase in post-vaccination titres, 95%.

The analysis was performed with the SAS software, using version 9.2 (SAS Institute, Cary, North Carolina, USA). For each antigen, a non-inferiority test was performed using the 95% two-sided confidence interval (CI) of the difference in the seroprotection rates between those receiving DTaP-IPV//Hib concomitantly with the second CYD-TDV dose and DTaP-IPV//Hib coadministered with placebo (a=2.5%>). The two-sided 95%> CI was calculated based on the Wilson score method without continuity correction as described by Newcombe R. (Stat Med., 1998; 17: 873-890). For each antigen, non-inferiority was demonstrated if the lower limit of the two-sided 95% CI was greater than -10%.

The per-protocol analysis dataset was used for the primary objective and confirmed in the full analysis set. The per-protocol analysis data set was defined as all participants who had no protocol deviations that could impact the DTaP-IPV//Hib vaccine immunogenicity up to Month 7. The full analysis set consisted of all participants who received at least the co-administration of DTaP-IPV//Hib vaccine with either the second dose of CYD-TDV or placebo, and who had a blood sample pre- and post-DTaP-IPV//Hib vaccination drawn, and a result available for at least one combination antigen (i.e., participants with at least one DTaP-IPV//Hib antigen value available at Month 6 or Month 7). The dengue immunogenicity analysis set consisted of participants randomized into the dengue immunogenicity subset who received at least one dose of CYD-TDV. The viremia analysis set, consisted of any participant included in the viremia analysis subset who received the first CYD-TDV injection, and provided at least one blood sample for which viremia laboratory results were available. The safety analyses were performed using the safety analysis set, defined as those participants who received at least one dose of CYD-TDV, DTaP-IPV//Hib vaccine, or placebo.

For the main parameters, 95% CIs of point estimates were calculated using the normal approximation for quantitative data and the exact binomial distribution (Clopper-Pearson method, quoted by Newcombe R. (Stat Med. 1998; 17: 857-872) for proportions. For immunogenicity, assuming that Logio transformation of the titers/data follows a normal distribution, the mean and the 95% CI were calculated on Logio (titers/data) using the usual calculation for normal distribution (using Student's t distribution with n-1 degrees of freedom), then anti-log transformations were applied to the results of calculations, in order to provide geometric mean of titers (GMTs) and geometric mean of titers ratio (GMTRs) and their 95% CI. There were no hypotheses tested for the secondary or additional objectives; all analyses were descriptive.

Results

A total of 720 participants were enrolled, of whom 309 were randomized to Group 1 and 315 to Group 2. The flow of participants through the study is shown in Figure 1. Overall, 129 (17.9%)) discontinued and the most frequently reported reason for discontinuation was voluntary withdrawal not due to an AE (77 participants [10.7%] including 58 participants that were not randomized), followed by non-compliance with the protocol (26 participants [3.6%] including 21 participants that were not randomized). Participant characteristics at baseline are summarized in Table 1.

Table 1

Group 1 Group 2 All

(N=301) (N=302) (N=603)

Sex: n (%)

Male 160 (53.2) 169 (56.0) 329 (54.6)

Female 141 (46.8) 133 (44.0) 274 (45.4)

Missing 0 (0.0) 0 (0.0) 0 (0.0)

Sex ratio: male/female 1.13 1.27 1.20

Age (months)

Mean (SD) 10.7 (1.12) 10.7 (1.17) 10.7 (1.14)

Min; Max 9.03; 13.0 9.03; 13.0 9.03; 13.0

Age group (months)

9-1 1 254 (84.4) 253 (83.8) 507 (84.1)

12 47 (15.6) 49 (16.2) 96 (15.9)

Weight (kg) Group 1 Group 2 All

(N=301) (N=302) (N=603)

Mean (SD) 9.20 (1.06) 9.18 (1.16) 9.19 (1.11)

Min; Max 6.20; 12.3 5.70; 14.2 5.70; 14.2

Height (cm)

Mean (SD) 72.9 (3.79) 72.6 (3.72) 72.7 (3.75)

Min; Max 65.0; 87.0 64.0; 89.0 64.0; 89.0

Racial origin

Asian 0 (0.0) 0 (0.0) 0 (0.0)

Black 0 (0.0) 0 (0.0) 0 (0.0)

Caucasian 0 (0.0) 0 (0.0) 0 (0.0)

Hispanic 245 (81.4) 249 (82.5) 494 (81.9)

American Indian or Alaska Native 0 (0.0) 0 (0.0) 0 (0.0)

Native Hawaiian or other Pacific Islander 0 (0.0) 0 (0.0) 0 (0.0)

Other 56 (18.6) 53 (17.5) 109 (18.1)

Dengue status

Immune 24 (8.0) 34 (11.3) 58 (9.6)

Non-immune 276 (91.7) 268 (88.7) 544 (90.2)

Missing 1 (0.3) 0 (0.0) 1 (0.2)

SD, standard deviation

Of the 719 participants with available data, 713 (99.2%) had not received yellow fever vaccination and had no history of yellow fever infection at baseline, but this data was missing for 6 (0.8%>) participants.

Non-inferiority of DTaP-IPV//Hib booster vaccine co-administered with CYD-TDV

The antibody response (28 days after vaccine administration) against all antigens (diphtheria, tetanus, pertussis, polio, and Hib) in participants who received DTaP- IPV//Hib booster vaccine concomitantly with the second dose of CYD-TDV was non- inferior to the antibody responses against all antigens following administration of DTaP- IPV//Hib booster vaccine and placebo (Table 2).

The lower limit of all of the 2-sided 95% CIs for the difference between the seroprotection/booster rates was greater than -10% for all of the antigens. The range for the lower limit of the differences in the 95% CIs was from -4.87 (for anti-FHA) to 1.14 (for anti-tetanus and anti-polio 1). The proportion of participants with seroprotection against diphtheria toxoid, tetanus toxoid, polio viruses types 1, 2, and 3, and PRP was 100% in Group 1 and ranged from 99.6% to 100% in Group 2. The proportion of participants with a booster response to PT and FHA was approximately 97% and 93%, respectively, in both groups.

CYD-TDV immunogenicity

The dengue immunogenicity subset included 109 participants in Group 1 and

107 participants in to Group 2. Concomitant DTaP-IPV//Hib booster vaccine administration with the second dose of CYD-TDV vaccine did not have any effect on seropositivity rates after the second and third dose of CYD-TDV. In Group 1, 28 days following the second CYD-TDV dose, the proportion of participants with antibody titers >10 (1/dil) were 84.8%, 98.1%, 100% and 90.5% for serotypes 1, 2, 3 and 4, respectively. The corresponding proportion of participants with dengue antibody titers >10 (1/dil) in Group 2 after the second CYD-TDV dose were 92.2%, 96.1%, 98.1% and 96.1%, respectively. After the third CYD-TDV dose, 100% of participants in both treatment groups were seropositive for all four dengue serotypes. GMTs of the antibodies against parental dengue virus serotypes in the two study groups, 28 days after the second and third CYD-TDV dose, are summarized in Figure 2. CYD-TDV viremia

The viremia subset included 45 participants in Group 1 and 47 participants in Group 2. Eight days after the first dose of CYD dengue vaccine, 32 participants (33.3%) had detectable (> lower limit of detection [LLOD]) non-serotype specific vaccine viremia: 13 (28.9%) participants in Group 1, 18 (38.3%) in Group 2 and one (25.0%>) who was not randomized at month 6.

Safety evaluations

There were no immediate unsolicited AEs following any injection. The safety and reactogenicity data up to 28 days after any injections are summarized in Table 3.

Grade 3 solicited reactions occurred at similar frequencies in Group 1 (18.8%) and Group 2 (16.2%). Five participants (1.6%) in Group 1 and four (1.3%) in Group 2 experienced at least one Grade 3 solicited injection site reaction, and 55 (17.8%) in Group 1 and 48 (15.2%) in Group 2 experienced at least one Grade 3 solicited systemic reaction.

Concomitant administration of DTaP-IPV//Hib booster vaccine with the second CYD-TDV dose did not have an impact on solicited systemic reactions compared with DTaP-IPV//Hib booster vaccine and placebo. The proportion of participants who reported at least one solicited systemic reaction within 14 days after vaccine injections at month 6 was 60.1% after concomitant administration of DTaP-IPV//Hib booster vaccine with the second CYD-TDV dose and 55.6% with DTaP-IPV//Hib booster vaccine and placebo. The proportion of participants reporting each of the solicited systemic reactions were similar in both groups and ranged from 11.7% to 37.7% in Group 1, and 11.8% to 39.7% in Group 2.

Overall, a total of 41 (5.7%) participants experienced at least one SAE at any time during the study: 17 (5.5%) in Group 1, 21 (6.7%) in Group 2, and 3 (3.3%) of those who were not randomized at month 6. None of these SAEs were considered related to the study vaccine by the Investigator. However, three SAEs (febrile seizures) were considered related to the study vaccine(s) by the Sponsor. One occurred one day after CYD-TDV and DTaP-IPV//Hib booster vaccine injection in a 17-month-old participant in Group 1, who had no history of seizures. Another occurred one day after the second dose of CYD-TDV in a 16-month-old participant in Group 2, with a family history of seizures. The third occurred 36 hours after the first dose of CYD-TDV in a 9-month-old participant (not randomized at month 6) with no history of seizure. No deaths occurred within 28 days of any injection. However, two non-related deaths (complex congenital cyanogen cardiopathy and post-operative complications in a participant not-randomized at month 6, and myelomonocytic leukemia in a participant in Group 2) were reported during the study. One participant in Group 1 experienced a SAE of special interest that was also considered significant (hemorrhagic dengue requiring hospitalization). Five participants reported non- serious AEs of special interest: 2 (0.6%) participants in Group 1 and 3 (1.0%) participants in Group 2. Only one episode, urticaria, was considered related to the study vaccines.

Safety Follow-up

Following the end of the active phase of the trial, an ongoing safety follow-up was carried out at the Merida (Yucatan) site. The follow-up is due to end in May 2017. During this follow-up period, up to the time of filing, no dengue case or serious dengue case has been identified.

Discussion

This study demonstrated the non-inferiority of immunological responses to

DTaP-IPV//Hib booster vaccine when concomitantly administered with the second CYD- TDV dose compared with concomitant administration with placebo. Both vaccines were immunogenic and well tolerated when concomitantly administered. Seroprotection rates against diphtheria, tetanus, pertussis, polio and Haemophilus influenzae antigens observed in the current study were within the range normally observed after administration of a booster dose of DTaP-IPV//Hib vaccine in toddlers (Plotkin S, et al, Expert Rev Vaccines. 2011; 10: 981-1005; Thisyakorn U, et al, Southeast Asian J Trap Med Public Health. 2009; 40: 282-294). Likewise, DTaP-IPV//Hib booster vaccination co-administered with the second dose of CYD-TDV did not affect the immunogenicity of the CYD-TDV as a 3- dose primary vaccination course.

GMTs reached following the third CYD-TDV dose were broadly consistent with those in another study in toddlers in the Philippines (CYD08 clinical trial - https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-001694-14/3rd; published as "First Experience of Concomitant Vaccination Against Dengue and MMR in Toddlers"; Crevat et al, Ped. Inf. Dis. J., (2015); Aug.; v.34(8): p.884-92; DOI No. 10.1097/INF.0000000000000752). There were no safety concerns following concomitant administration of the second CYD-TDV dose with the DTaP-IPV//Hib booster vaccination. No immediate reactions were reported, and unsolicited adverse reactions occurred in less than 1% of the participants. SAEs occurred at a similar frequency in both study groups. Three SAEs (febrile seizures; one in Group 1, one in Group 2, and one in the group not randomized) were considered by the study Sponsor as possibly related to study vaccinations. However, a postlicensure safety surveillance study has previously found seizures to be rare following administration of the DTaP-IPV//Hib vaccine (Nelson JC et al., Am J Epidemiol. 2013; 177: 131-141). Very few participants (< 1%) discontinued due to adverse events. The safety profile of the DTaP-IPV//Hib vaccine in the current study was broadly consistent with that observed in other phase III studies conducted in toddlers with a booster dose of this vaccine, although irritability was slightly more frequent in both treatment groups in the current study (Plotkin S et al, Expert Rev Vaccines. 2011; 10: 981-1005). Following administration of CYD-TDV the frequency of injection site reactions and systemic reactions appears to be greater in the current study compared with a previous phase II trial conducted in toddlers (CYD08).

One dengue case was reported during the study; a 19-month-old female participant from Group 1 developed hemorrhagic dengue 113 days after receiving the study vaccines (DTaP-IPV//Hib booster vaccination co-administered with the second dose of CYD-TDV). The child developed hyperthermia and was subsequently hospitalized. The case was assessed as non-severe and the child fully recovered. The event was assessed by both the Investigator and the Sponsor as not related to the study vaccine.

The current study has a number of limitations. Although participants in group 2 received three doses of CYD-TDV, the schedule used (0, 7 and 12 months) was different to the schedule (0, 6 and 12 months) used in group 1 and in Phase III clinical trials. Nonetheless, the GMTs of the antibodies against parental dengue virus serotypes in the two study groups, 28 days after the second and third CYD-TDV dose were similar.

In conclusion, the second dose of CYD-TDV can be concomitantly administered with the DTaP-IPV//Hib booster vaccination without significant impact on immunogenicity and safety of the DTaP-IPV//Hib vaccine. Also, the DTaP-IPV//Hib booster vaccination can be concomitantly administered with the second dose of CYD-TDV without significant impact on immunogenicity and safety of the CYD-TDV vaccine.

o o

Table 2: Non-inferiority ofDTaP_rIPV H|b vaccine booster dose based on seiogroteetion booster response 28 days after injection - Per Protocol Analysis Set

Booster response (based on pre-dose blood sample {month 6)): pre-dose titer is < LLOQ and thepost-do e titer is = 4 x LLOQ or the pie-dose titer is = 1XOQ but< 4 x LLOQ andfhepost-dose titer is = 4 fold-rise or the pre-dose Uteris. = 4 x LLOQ and the post-dose titer is = 2 fold-rise

Ifpre or post-dose titer is missing., thentheboosterresponse is missing.

Ex a ct binomial metho d (gkjjjjjejr-Pe arson memo d, quoted by ¾e_¾y¾mbg ) used for the single proportion 95% two sided C Is.

Ihe non-inferiority will be demonstrated if the lower limit of all the 95% CI of the difference is greater than -10% for all antigens.

U number of participants experiencing the endp oint list ed in the first three columns

M: num er of participants with available data for the relevant endpoint

LLOQ, lower limit of quantification; PT_jgjggjjggjs. |ojggjd PRP,

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TaMejL Safely overview within 28 days after any injections (Safety Analysis Set^(sonrce Table 6.1))|

Group 1 Group 2 Not rinioinized it month 6

{N=309) (N-315)

nJM % (95% CI) n/M W <95<* CI) n/M % (SS% CI)

Solicited reaction 284/309 91.9 (883: 94.7) 284/315 902 {86.3 93.2) 47/67 70.1 {57.7; 80.7)

Solicited injection site reaction 191/309 61.8 (56.1 ; 67.3) 198/315 62.9 {57.3 68.2) 22/65 33.S (22.6; 46.6)

a fter injection with DTaP-IPW/Hib 1 18/301 39.2 (33.7; 45.0) 125/306 40.8 {35.3 46.6) NA ^'NA NA

a ter injection with CYD-TDV 171/309 55.3 (49.6; 61.0) 165/315 52.4 {46.7 58.0) 22/65 33.8 (22.6; 46.6)

a fter injection with pla cebo 73 294 24.8 (20.0; 30.2) 108/306 35.3 {29.9 40.9) NA NA NA

Solicited systemic reaction 264/309 85.4 (81.0: 89.2) 271/315 86.0 {81.7 89.7) 43/67 64.2 {51.5; 75.5)

Unsolicited AE 225/309 72.8 (67.5; 77.7) 239/ 15 75.9 {70.8 80.5) 26/90 28.9 {19.8; 39.4)

Unsolicited AR 2/309 0.6 (0.1 ; 2.3) 4/315 1.3 {0.3 3.2) 0/90 0.0 (0.0; 4.0)

Unsolicited non-serious AE 223/309 72.2 (€6.8; 77.1) 238/315 75.6 {70.4 80.2) 25/90 27.8 {18.9; 38.2)

Unsolicited non-serious AR 2/309 0.6 {0.1 ; 2.3) 4/315 1.3 {0.3 3.2) 0/90 0.0 {0.0; 4.0)

Unsolicited non-serious injection site AR 1/309 0.3 (0.0; 1.8) 4/315 1.3 {0.3 3.2) 0/90 0.0 (0.0; 4.0)

a ft er injection with DTaP-IPV' Hib 0/309 0.0 (0.0; 1.2) 1/315 0.3 {0.0 1.8) NA NA NA

a fter injection with CYD-TDV 1/309 0.3 (0.0; 1.8) 3/315 1.0 {0.2 2.8) 0/90 0.0 {0.0; 4.0)

a fter injection o f laceb o 0/309 0.0 (0.0: 1.2) 0/315 0.0 {0.0 1.2) NA NA NA

Unsolicited non-serious systemic AE 223/309 72.2 {66.8; 77.1) 238/315 75.6 {70.4 80.2) 25/90 27.8 (18.9; 38.2)

Unsolicited non-serious systemic AR 1/309 0.3 {0.0: 1.8) 0/315 0.0 {0.0 1.2) 0/90 0.0 (0.0; 4.0)

AE leading to study d-scontinuition* 0/309 0.0 {0.0; 1.2) 0/315 0.0 {0.0 1.2) 1/90 1.1 {0.0; 6.0)

SAE 1 1/309 3.6 {1.8; 6.3) 7/315 2.2 {0.9 4.5) 1/90 1.1 {0.0; 6.0)

Death 0/309 0.0 {0.0; 1.2) 0/315 0.0 (0.0 12) 0/90 0.0 {0.0; 4.0)

n: number of participants experiencing the endpoint listed in the first column; M: number of participants with available data for the relevant endpoint

*AEs include serious AEs

AE, a CI. confidence interval; CYD-TDV, tetravalent dengue vaccine; JK&J-IIWjljJj, diphtheria, tetanus. .ge ii j; pertussis, in cti ated olio

not applicable; SAE, serious adverse event

Sequence Listing

SEQ ID N° Corresponding definitions

1 prM+E nucleotide sequence of a serotype 1 dengue fever strain

2 prM+E nucleotide sequence of a serotype 2 dengue fever strain

3 prM+E nucleotide sequence of a serotype 3 dengue fever strain

4 prM+E nucleotide sequence of a serotype 4 dengue fever strain prM+E nucleotide sequence of a serotype 2 dengue fever strain

5

(CYD-2V)

6 Entire nucleotide sequence of VDV1

7 Entire nucleotide sequence of VDV2

Claims

1. A combined DTaP-IPV/Hib vaccine for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio, and Haemophilus influenzae type b, wherein said method comprises concomitantly administering said DTaP-IPV/Hib vaccine to a human subject together with a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4.

2. The combined DTaP-IPV/Hib vaccine for use in a method according to claim 1, wherein the dengue vaccine is administered according to a three-dose dengue vaccination schedule.

3. The combined DTaP-IPV/Hib vaccine for use in a method according to claim 2, wherein the second dose of said dengue vaccine is concomitantly administered with the DTaP-IPV/Hib vaccine.

4. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 1 to 3, wherein the DTaP-IPV/Hib vaccine comprises a mixture of antigens for protection against diphtheria, tetanus, whooping cough (pertussis), polio and infections caused by Haemophilus influenzae type b.

5. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 1 to 4, wherein the DTaP-IPV/Hib vaccine comprises a diphtheria toxoid; a tetanus toxoid; an acellular pertussis antigen; an inactivated poliovirus (IPV) and a Haemophilus influenza type b capsular polysaccharide covalently bound to tetanus toxoid.

6. The combined DTaP-IPV/Hib vaccine for use in a method according to claim 5, wherein the acellular pertussis antigen is selected from the group consisting of a detoxified pertussis toxoid, a filamentous haemagglutinin or a combination thereof.

7. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 5 to 6, wherein the Haemophilus influenza type b capsular polysaccharide is polyribosylribitol phosphate.

8. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 5 to 7, which comprises an inactivated poliovirus (IPV) of Type 1 , Type

2 and Type 3.

9. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 1 to 8, wherein the dengue antigens of serotypes 1, 3 and 4 are each a live attenuated chimeric dengue virus, and the dengue antigen of serotype 2 is selected from the group consisting of a live attenuated dengue virus and a live attenuated chimeric dengue virus.

10. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 1 to 8, wherein the dengue antigens of serotypes 1, 3 and 4 are each independently selected from the group consisting of a live attenuated dengue virus and a live attenuated chimeric dengue virus, and the dengue antigen of serotype 2 is a live attenuated chimeric dengue virus.

11. The combined DTaP-IPV/Hib vaccine for use in a method according to claim 9 or claim 10, wherein the dengue antigens of serotypes 1 to 4 are each a live attenuated chimeric dengue virus.

12. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 9 to 11, wherein the live attenuated chimeric dengue virus comprises an envelope (E) protein from a dengue virus and one or more proteins from an attenuated yellow fever virus.

13. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 9 to 12, wherein the live attenuated chimeric dengue virus comprises a genome of an attenuated yellow fever virus whose the prM-E sequence has been substituted with the prM-E sequence of a dengue virus.

14. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 1 to 13, wherein the subject is dengue immune and/or the subject resides in a dengue endemic area.

15. The combined DTaP-IPV/Hib vaccine for use in a method according to any one of claims 1 to 14, wherein said method also results in the induction of a neutralizing antibody response against all four serotypes of dengue and wherein said neutralizing antibody response is non- inferior to the neutralizing antibody response against the four serotypes of dengue induced by sequential administration of said combined DTaP- IPV/Hib vaccine and said tetravalent dengue vaccine.

16. A tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4, for use in a method for inducing a protective immune response against diphtheria, tetanus, whooping cough (pertussis), polio and Haemophilus influenzae type b, wherein said method comprises concomitantly administering said dengue vaccine to a human subject together with a combined DTaP-IPV/Hib vaccine.

17. The dengue vaccine for use in a method according to claim 16, wherein the live attenuated dengue vaccine is administered according to a three-dose dengue vaccination schedule.

18. The dengue vaccine for use in a method according to claim 17, wherein the second dose of said dengue vaccine is concomitantly administered with the combined DTaP-IPV/Hib vaccine.

19. The dengue vaccine for use in a method according to claim 17 or claim 18, wherein the DTaP-IPV/Hib vaccine comprises a mixture of antigens for protection against diphtheria, tetanus, whooping cough (pertussis), polio and infections caused by Haemophilus influenza type b.

20. The dengue vaccine for use in a method according to any one of claims 17 to 19, wherein the combined DTaP-IPV/Hib vaccine comprises a diphtheria toxoid; a tetanus toxoid; an acellular pertussis antigen; an inactivated poliovirus (IPV) and a Haemophilus influenza type b capsular polysaccharide covalently bound to tetanus toxoid.

21. The dengue vaccine for use in a method according to claim 20, wherein the acellular pertussis antigen is selected from the group consisting of a detoxified pertussis toxoid, a filamentous haemagglutinin or a combination thereof.

22. The dengue vaccine for use in a method according to claim 20 or claim 21, wherein the Haemophilus influenza type b capsular polysaccharide is polyribosylribitol phosphate.

23. The dengue vaccine for use in a method according to any of claims 20 to 22, which comprises an inactivated poliovirus (IPV) of Type 1, Type 2 and Type 3.

24. The dengue vaccine for use in a method according to any one of claims 17 to 23, wherein the dengue antigens of serotypes 1, 3 and 4 are each a live attenuated chimeric dengue virus, and the dengue antigen of serotype 2 is selected from the group consisting of a live attenuated dengue virus and a live attenuated chimeric dengue virus.

25. The dengue vaccine for use in a method according to any one of claims 17 to 23, wherein the dengue antigens of serotypes 1, 3 and 4 are each independently selected from the group consisting of a live attenuated dengue virus and a live attenuated chimeric dengue virus, and the dengue antigen of serotype 2 is a live attenuated chimeric dengue virus.

26. The dengue vaccine for use in a method according to claim 24 or claim 25, wherein the dengue antigens of serotypes 1 to 4 are each a live attenuated chimeric dengue virus.

27. The dengue vaccine for use in a method according to any one of claims 24 to 26, wherein the live attenuated chimeric dengue virus comprises an envelope (E) protein from a dengue virus and one or more proteins from an attenuated yellow fever virus.

28. The dengue vaccine for use in a method according to any one of claims 24 to 27, wherein the live attenuated chimeric dengue virus comprises a genome of an attenuated yellow fever virus whose the prM-E sequence has been substituted with the prM-E sequence of a dengue virus.

29. The dengue vaccine for use in a method according to any one of claims 17 to 28, wherein the subject is dengue immune and/or the subject resides in a dengue endemic area.

30. The dengue vaccine for use in a method according to any one of claims 17 to 29, wherein said method also results in the induction of a neutralizing antibody response against all four serotypes of dengue and wherein said neutralizing antibody response is non-inferior to the neutralizing antibody response against the four serotypes of dengue induced by sequential administration of said combined DTaP-IPV/Hib vaccine and said tetravalent dengue vaccine.

31. A vaccine composition comprising a mixture of a combined DTaP- IPV/Hib vaccine and of a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4.

32. The vaccine composition according to claim 31, comprising a mixture of :

- a combined DTaP-IPV/Hib vaccine in an amount sufficient for inducing a protective immune response in a human subject, and

- a tetravalent dengue vaccine which comprises a live attenuated dengue virus of each of serotypes 1 to 4 in an amount sufficient for a second dose of a dengue vaccine adapted for a three-dose dengue vaccination schedule in a human subject.

33. The vaccine composition according to claim 31 or claim 32, wherein the tetravalent dengue vaccine which comprises a live attenuated chimeric dengue virus of each of serotypes 1 to 4, wherein the live attenuated chimeric dengue virus comprises an envelope (E) protein from a dengue virus and one or more proteins from an attenuated yellow fever virus.