Highly neutralizing antibody responses for protective immunity against influenza infections have been associated with the hemagglutinin (HA) glycoprotein. Therefore, this protein has been a major focus for therapeutic intervention in influenza infections. Most of the influenza vaccines target this protein to induce immune responses in the host, mainly in the form of neutralizing antibody based response 
. However, whenever immediate protection becomes essential and there is no time to induce an antibody response, the best alternative so far is passive immunization. Also, since drug resistant strains of H5N1 viruses are emergent, it is vital to explore other means of therapy for H5N1 infections 
. Passive monoclonal antibody based therapy is a viable option that can be investigated.
Previous studies have proven the efficacy of humanized and human monoclonal antibodies as therapy in murine models of H5N1 infection 
. However, these studies only discuss the application of single monoclonal antibodies against infections with some strains of H5N1 virus. A single monoclonal antibody may not be sufficient to protect against all circulating strains 
. Also, utilizing a single mAb against one epitope can result in selective pressure-induced “escape” of the virus through point mutations that can alter antibody binding 
. The above mentioned studies with H5N1 infections have not looked into this aspect in great detail. In view of the proven efficiency of passive prophylaxis and therapy of H5N1 infection in animal models using single mAbs, we evaluated the prospect of using a combination of monoclonal antibodies to tackle the issues posed by using single mAbs as therapy. As is evident from our studies, the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy against H5N1 infection.
We chose the mAbs based on their recognition of non-overlapping and non-competing epitopes. In combination, mAbs 2D9 and 4C2 could neutralize all of the strains from phylogenetically distinct clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8. Also, the escape mutants generated from each of these mAbs could be efficiently neutralized by the other. We deduced that the combination of these mAbs would be very efficient in therapy against most strains of H5N1. Hence, we used the combination of these mAbs for prophylaxis and therapy against H5N1 in a mouse model of infection.
The results of the present study demonstrate that passive administration of a combination of two different neutralizing chimeric mAbs against HA1 can effectively protect against highly pathogenic H5N1 infection, when administered either as prophylaxis or therapeutics. We observed that 10 mg/kg of ch-mAb was required for the protection of 100% of the mice when a single mAb was used. However, when the combination of mAbs was used under the same conditions of viral challenge, only 5 mg/kg of a single dose or 2.5 mg/kg of the double dose was needed to offer complete protection. Administration of two doses of the combination showed better protection as the viral loads in the lungs were significantly reduced when compared to administration of a single dose. Moreover, a double dose of the combination of mAbs controlled immune escape as no escape mutants were isolated from the lungs of the groups of mice treated at 48 h intervals with two doses.
We observed the generation of escape mutants in vivo in 100% of the cases whenever a single monoclonal antibody was used for therapy (data not shown). However, using two monoclonal antibodies in combination also showed the generation of escape mutants whenever sub-neutralizing concentrations of mAbs were used. Using higher concentrations in a single dose (5 mg/kg) reduced this possibility. This may have been dependent on the amount of the complementary circulating mAb present in the system which could effectively neutralize the escape mutants due to any one of the mAb. Based on the evidence from the body weight of the mice, the mice were healthy enough from the single administration for the escape mutants to be cleared by the active immune system of the mice. Given that the half life of the mAbs is limited, it is evident as to why two doses of the mAb combination worked in a much better way and provided better safety against the emergence of escape variants. The high rate of emergence of escape mutants to these viruses are evidence that the antibodies are highly neutralizing and hence forcing the viruses to adapt. But the fact that using both ch-mAbs in combination in two doses did not give rise to any escape mutants is proof that the antibodies are complementary and hence offer complete protection to the mice.
Previous studies with other viruses have shown that combination of two or more than two mAbs directed against different epitopes could lead to a two to ten fold increase in neutralization titers 
and provided greater protection against many other diseases 
. Moreover, Meulen et al. 
reported that much better control of potential neutralization escape variants could be achieved with an antibody combination against SARS Coronavirus. However, no studies have been done so far to demonstrate the efficacy of combination therapy against H5N1 infection.
Though the dose of antibodies delivered for complete protection of mice was quite high, we believe that further improvement of these antibodies as well as their inclusion in an antibody cocktail will ensure better protection. Our data provide a rationale to develop combinations of mAbs for human H5N1 prophylaxis and therapeutics. The combination of two mAbs expanded the breadth of protection with a high level of efficacy and safety associated with potential immune escape variants. Also, combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak. In future, it may be possible to generate humanized monoclonal antibodies of mAb 2D9 and 4C2 by CDR (Complementarity Determining Regions) grafting and further facilitate their use in non-primate and human clinical trials.