Immunity to Influenza in Ferrets: II. Influence of Adjuvants on Immunization

Immunization of ferrets with a single intramuscular inoculation of killed A2/Hong Kong virus did not induce serum or nasal antibody, and these animals were found to be completely susceptible to subsequent infection with virulent influenza virus A2/Hong Kong/3/68. A similar result was found for ferrets immunized with 2 inoculations of killed virus vaccine given 2 weeks apart. Ferrets immunized with killed A2/Hong Kong virus in conjunction with Bordetella pertussis produced relatively low levels of serum HI antibody to A2/Hong Kong virus; when infected with virulent influenza virus, these ferrets showed a modified reaction, with a less marked febrile reaction than was observed for non-immunized animals.Immunization of ferrets with killed A2/Hong Kong virus in Freund''s complete adjuvant resulted in the production of relatively high levels of serum HI antibody, but no detectable nasal antibody. These animals were shown to be partially immune to subsequent infection with virulent influenza virus. However, although the serum antibody levels of these animals following immunization was comparable to that found following infection with live virus, the degree of immunity to infection with virulent influenza virus was measurably less.     

Immunity to influenza in Ferrets

Summary Ferrets inoculated with 300 CCA of inactivated influenza A2/Hong Kong virus vaccine did not produce serum HI antibody, and were completely susceptible to subsequent infection with live A2/Hong Kong virus. Immunization of ferrets with A2/Hong Kong vaccine in Al(OH)₃ induced low levels of serum HI antibody; these animals showed a slightly reduced febrile reaction and reduced titres of virus were recovered from nasal washings following challenge virus infection. Ferrets immunized with inactivated A2/Hong Kong vaccine in Freund's incomplete adjuvant produced relatively high titres of serum HI antibody, but did not produce local antibody detectable in nasal washings. After challenge infection, these animals showed a modified febrile reaction, lower titres of virus were recovered from nasal washings and nasal symptoms were reduced. These results, together with results of similar studies, indicated that the degree of immunity to challenge virus infection was related to the titre of serum HI antibody. However, none of the methods used to induce serum HI antibody gave as solid an immunity as found following live virus infection, although immunization could induce levels of serum HI antibody comparable to that found following virus infection.     

Immunity to Influenza in Ferrets: I. Response to Live and Killed Virus

Ferrets were found to react with a sharp febrile response to intranasal infection with influenza virus A2/Hong Kong/3/68. Virus was recovered from nasal washings taken 3 days after infection, and virus antibody was found in serum specimens taken 21 days after virus infection. Virus infection produced a pronounced rhinitis; the protein concentration in nasal washings was found to increase three to five-fold with peak levels occurring on day 7, post-infection. Concomitant with the increased protein levels, detectable levels of HI and neutralizing antibody were found in the nasal washings. However, nasal washings taken 13 days or more after influenza virus infection did not contain either increased levels of protein or detectable antibody. These ferrets were immune to re-infection with homologous virus inoculated 5 weeks after primary infection. Thus, ferrets showed no febrile response; virus was not recovered from nasal washings; serum antibody titres did not increase; no increase in protein levels was found in nasal washings; and HI antibody was not found in nasal washings.Using these criteria to assess susceptibility or immunity to influenza virus infection, infection with attenuated influenza virus A2/Hong Kong/1/68 produced immunity to re-infection with virulent virus. Ferrets infected with influenza virus B/England/13/65 or immunized with killed A2/Hong Kong virus did not induce any immunity to infection with influenza virus A2/Hong Kong/3/68.     

Immunity to influenza virus infection induced by heterologous,inactivated vaccines

The ability of inactivated influenza A vaccines to induce serum HI antibody and immunity to challenge infection was studied in hamsters and in volunteers. Groups of hamsters were immunized with 200 IU of influenza virus A/Scotland/74, A/Port Chalmers/73, A/England/72, or A/Hong Kong/68. The serum HI antibody response of animals to, and immunity to challenge infection was directly related to the known relationship between the vaccine and test viruses. Thus, hamsters given A/Hong Kong/68 or A/England/72 vaccine produced serum HI antibody and immunity to A/Hong Kong virus infection, and animals given A/Scotland/74, A/Port Chalmers/73, and A/England/72 produced antibody and immunity to A/Scotland infection.In a volunteer study, groups of students were immunized with 400 IU of the same vaccines as used above. The ability to infect these volunteers with WRL 105 virus given 4 weeks later was directly related to the vaccine-induced serum HI antibody to the challenge virus. The highest titers of serum HI antibody to A/Scotland virus were found in volunteers inoculated with homologous vaccine, lower titers were found in volunteers given A/Port Chalmers or A/England/ 72 vaccine and the lowest levels were seen in volunteers given A/Hong Kong/68 vaccine: the largest number of infections by the challenge virus was seen in volunteers given A/Hong Kong/68 vaccine, less were observed in volunteers given A/England/72 vaccine, and least were found in groups given A/Port Chalmers or A/Scotland/74 vaccine. Compared with the incidence of infection in volunteers given B/Hong Kong/73 vaccine, all groups given heterologous influenza A vaccines showed some immunity to challenge infection.     

Immunity to influenza in ferrets

Ferrets were infected with recombinant influenza A viruses which possessed either the haemagglutinin or neuraminidase antigens of A/Hong Kong/68 influenza virus. After five weeks the immunity of the animals was challenged by infection with A/HK/68 virus. Immunity to challenge infection was greatest in those ferrets with serum HI antibody to A/HK/68; the presence of NI antibody conferred a measurably lower degree of immunity. A small degree of heterotypic immunity was observed following challenge infection of ferrets previously infected with influenza virus A/PR/8/34, although the surface antigens of this virus are completely different from those of A/HK/68. Experiments in which ferrets were infected with A/HK/68 virus and subsequently challenged with the recombinant viruses confirmed the results of the first experiment.     

Influenza virus infection of the guinea pig: Immune response and resistance

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.     

Failure of attenuated temperature-sensitive influenza A (H3N2) virus to induce heterologous interference in humans to parainfluenza type 1 virus.

The present investigation was undertaken to determine if a candidate live vaccine virus, influenza A/Hong Kong/68-ts-1 [E] (H3N2), induced heterologous interference against an interferon-sensitive, wild-type, parainfluenza type 1 challenge virus. The parainfluenza virus was administered 7 days after Hong Kong/68-ts-1 [E] virus infection. The clinical response, daily quantitative virus shedding, interferon production, and serum and nasal wash antibody responses were determined in an experimental group (influenza A virus followed by parainfluenza virus) and 10 volunteers in a control group (parainfluenza virus only). The volunteers were selected on the basis of susceptibility to the two viruses, i.e. serum hemagglutination-inhibition antibody titer of is less than or greater to 1:8 for influenza virus and low nasal wash antibody titer (is less than or greater to 1:8) for parainfluenza virus. Despite a 100% infection rate in the Hong Kong/68-ts-1 [E] vaccinees, no heterologous interference was induced against the parainfluenza type 1 virus challenge.     

The Local Origin of the Febrile Response Induced in Ferrets During Respiratory Infection with a Virulent Influenza Virus

Intranasal infection of ferrets with a virulent Clone (7a) of the recombinant influenza virus A/PR/8/34—A/England/939/69 (H₃N₂) produced a fever approximately 24 h in duration beginning about 29 h after infection. The origin of this fever has been investigated as an indication of what might happen in influenza in man.The systemic production of fever by virus interaction with phagocytes in the reticuloendothelial system appeared unlikely because insufficient virus escaped into the bloodstream. Ten half-hourly i.v. injections of 10⁸ 50%0 Egg-Bit Infectious Doses (EBID₅₀) of virus were needed to produce a fever of short duration (3-8 h). Yet, after the intranasal infection, which results in the 24 h fever, the total virus content in the nasal mucosa was less than 10⁸ EBID₅₀ before the onset of fever and only reached 10^8.5 EBID₅₀ for 4 h during fever. Also, just before or during the fever produced by intranasal infection, influenza virus antigens could not be detected by fluorescent antibody in the spleens of the animals but were detected in animals receiving a single bloodstream injection of 10⁸ EBID₅₀ of virus.Fever is more likely to result from release of leucocyte pyrogen by virus-phagocyte interaction in the upper respiratory tract. A pyrogen active in ferrets with the characteristics of leucocyte (endogenous) pyrogen was produced by incubating influenza virus with ferret peripheral phagocytes in vitro. A pyrogen with similar properties was released by incubation of nasal inflammatory cells collected from infected febrile ferrets and many of the cells were shown by fluorescent antibody to have interacted with influenza virus.     

Virus-like particle vaccine containing hemagglutinin confers protection against 2009 H1N1 pandemic influenza

Immunization of the world population before an influenza pandemic such as the 2009 H1N1 virus spreads globally is not possible with current vaccine production platforms. New influenza vaccine technologies, such as virus-like-particles (VLPs), offer a promising alternative. Here, we tested the immunogenicity and protective efficacy of a VLP vaccine containing hemagglutinin (HA) and M1 from the 2009 pandemic H1N1 influenza virus (H1N1pdm) in ferrets and compared intramuscular (i.m.) and intranasal (i.n.) routes of immunization. Vaccination of ferrets with VLPs containing the M1 and HA proteins from A/California/04/2009 (H1N1pdm) induced high antibody titers and conferred significant protection against virus challenge. VLP-vaccinated animals lost less weight, shed less virus in nasal washes, and had markedly lower virus titers in all organs tested than naïve controls. A single dose of VLPs, either i.m. or i.n., induced higher levels of antibody than did two doses of commercial split vaccine. Ferrets vaccinated with split vaccine were incompletely protected against challenge; these animals had lower virus titers in olfactory bulbs, tonsils, and intestines, but lost weight and shed virus in nasal washes to a similar extent as naïve controls. Challenge with heterologous A/Brisbane/59/07 (H1N1) virus revealed that the VLPs conferred minimal cross-protection to heterologous infection, as revealed by the lack of reduction in nasal wash and lung virus titers and slightly higher weight loss relative to controls. In summary, these experiments demonstrate the strong immunogenicity and protective efficacy of VLPs compared to the split vaccine and show that i.n. vaccination with VLPs has the potential for highly efficacious vaccination against influenza.     

Virus-like particle vaccine conferred complete protection against a lethal influenza virus challenge

We have previously demonstrated the formation and release of influenza virus-like particles (VLPs) from the surface of Sf9 cells infected with either a quadruple baculovirus recombinant that simultaneously expresses the influenza structural proteins hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), and matrix 2 (M2), or a combination of single recombinants that include the M1 protein. In this work, we present data on the immunogenicity and protective efficacy afforded by VLPs (formed by M1 and HA) after immunization of mice. VLP vaccine ( approximately 1 microg HA) were formulated with or without IL-12 as adjuvant and administered twice, at 2-week intervals, by either intranasal instillation or intramuscular injection. All VLP-vaccinated and influenza-immunized control mice demonstrated high antibody titers to the HA protein; however, intranasal instillation of VLPs elicited antibody titers that were higher than those induced by either intramuscular inoculation of VLPs or intranasal inoculation with two sub-lethal doses of the challenge influenza virus (control group). Antibody responses were enhanced when VLP vaccine was formulated with IL12 as adjuvant. All mice were challenged with 5 LD50 of a mouse-adapted influenza A/Hong Kong/68 (H3N2) virus. Intramuscular administration of VLP vaccine formulated with or without IL-12 afforded 100% protection against a lethal influenza virus challenge. Similarly, intranasal instillation of VLP vaccine alone protected 100% of the mice, whereas VLP formulated with IL-12 protected 90% of the vaccinated mice. Not only do these results suggest a novel approach to the development of VLP vaccines for diverse influenza virus strains, but also the creation of multivalent vaccines by decoration of the surface of the VLPs with antigens from other pathogens.     

Cross-Protection in Mice After Immunization with H2N2, H3N2, and Heq2Neq2 Influenza Virus Strains

Mice were vaccinated with the influenza viruses A/Japan/57 (H2N2), A/Hong Kong/68 (H3N2), and A/Equi/Miami/63 (Heq2Neq2) and the hemagglutinin and neuraminidase recombinants derived from these viruses. After infection with the parent viruses, protection was compared with serological findings. It was found that influenza vaccine protects not only against infection with a strain identical or closely related to the vaccine strain, but against heterologous strains as well. Vaccination with Hong Kong/68 and its neuraminidase recombinant resulted in a heterologous neuraminidase inhibition titer against Japan/57 and in a protection against infection with Japan/57. By contrast, after vaccination with Japan/57 and its neuraminidase recombinant, no relevant heterologous neuraminidase inhibition titer against Hong Kong/68 was observed, whereas a protection against infection with Hong Kong/68 did exist. A cross-protection between Hong Kong/68 and Miami/63, but no relationship in the hemagglutination or neuraminidase inhibition tests, was established in the preinfection sera. A one-way antigenic relationship between these viruses was confirmed by the rise of hemagglutinin or neuraminidase antibodies against Hong Kong/68 in the postinfection sera. No cross-protection or serological relationship existed between Miami/63 and Japan/57. Besides the hemagglutinin and neuraminidase, a third factor, the “mouse-protecting antigen,” was considered to contribute to the protection obtained. According to the protection observed, the mouse-protecting antigen of Hong Kong/68 virus is related to that of Japan/57 as well as Miami/63 virus. The mouse-protecting antigens of both Japan/57 and Miami/63 are related to that of Hong Kong/68.     

Immunization of Ferrets against Influenza: A Comparison of Killed Ferret Grown and Egg Grown Virus

Both killed unadjuvanted ferret and egg grown A/Moscow/1019/65 (H₂N₂) influenza virus failed to immunize ferrets against challenge with homologous virus; the preparations were given in 2 doses, 2 weeks apart, distributed intranasally, intramuscularly and intraperitoneally. However, small doses (<2 HA units) of both preparations induced immunity in ferrets previously “primed” with a live heterologous virus (A/FM/1/47 (H₁N₁)) according to the method of Potter et al. (1973a, b). Although no difference in immunizing activity was detected between ferret and egg grown virus, the former induced greater HI titres than the latter. There was no correlation between HI titres in serum and protection to challenge; in fact, some protection seemed to be afforded by the “priming” virus in the absence of HI antibody to the challenge virus.These results are discussed in relation to the possibility that a previously unrecognized antigen different from haemagglutinin and neuraminidase may contribute to immunity to influenza.     

Humans and Ferrets with Prior H1N1 Influenza Virus Infections Do Not Exhibit Evidence of Original Antigenic Sin after Infection or Vaccination with the 2009 Pandemic H1N1 Influenza Virus

The hypothesis of original antigenic sin (OAS) states that the imprint established by an individual''s first influenza virus infection governs the antibody response thereafter. Subsequent influenza virus infection results in an antibody response against the original infecting virus and an impaired immune response against the newer influenza virus. The purpose of our study was to seek evidence of OAS after infection or vaccination with the 2009 pandemic H1N1 (2009 pH1N1) virus in ferrets and humans previously infected with H1N1 viruses with various antigenic distances from the 2009 pH1N1 virus, including viruses from 1935 through 1999. In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1 virus-primed ferrets. Analysis of paired samples of human serum taken before and after vaccination with a monovalent inactivated 2009 pH1N1 vaccine showed a significantly greater-fold rise in the titer of antibody against the 2009 pH1N1 virus than against H1N1 viruses that circulated during the childhood of each subject. Thus, prior experience with H1N1 viruses did not result in an impairment of the antibody response against the 2009 pH1N1 vaccine. Our data from ferrets and humans suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus.     

In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group.     

Adeno-Associated Virus 9-Mediated Airway Expression of Antibody Protects Old and Immunodeficient Mice against Influenza Virus

Influenza causes serious and sometimes fatal disease in individuals at risk due to advanced age or immunodeficiencies. Despite progress in the development of seasonal influenza vaccines, vaccine efficacy in elderly and immunocompromised individuals remains low. We recently developed a passive immunization strategy using an adeno-associated virus (AAV) vector to deliver a neutralizing anti-influenza antibody at the site of infection, the nasal airways. Here we show that young, old, and immunodeficient (severe combined immunodeficient [SCID]) mice that were treated intranasally with AAV9 vector expressing a modified version of the broadly neutralizing anti-influenza antibody FI6 were protected and exhibited no signs of disease following an intranasal challenge with the mouse-adapted H1N1 influenza strain A/Puerto Rico/8/1934(H1N1) (PR8) (Mt. Sinai strain). Nonvaccinated mice succumbed to the PR8 challenge due to severe weight loss. We propose that airway-directed AAV9 passive immunization against airborne infectious agents may be beneficial in elderly and immunocompromised patients, for whom there still exists an unmet need for effective vaccination against influenza.     

Intranasal immunization of ferrets with commercial trivalent influenza vaccines formulated in a nanoemulsion-based adjuvant

NB-1008 is a surfactant-stabilized soybean oil-in-water nanoemulsion (NE) adjuvant with influenza virus antigen incorporated into the NE by simple mixing. Intranasal administration of the antigen with NE adjuvant efficiently produces both mucosal and serum antibody responses as well as a robust cellular Th1 immune response. To demonstrate the adjuvant effect of the W₈₀5EC NE, a killed commercial influenza vaccine for intramuscular administration (Fluzone or Fluvirin) was mixed with the W₈₀5EC NE adjuvant and administered intranasally to naïve ferrets. After a single intranasal immunization, the adjuvanted influenza vaccine elicited elevated serum hemagglutination inhibition (HAI) geometric mean titers (GMTs) ranging from 196 to 905 for the three hemagglutinin (HA) antigens present in the vaccine, which are approximately 19- to 90-fold higher titers at 1/50 the standard intramuscular commercial nonadjuvanted influenza vaccine dose. Seroconversion rates of 67% to 100% were achieved against each of the three viral strains present. The adjuvanted nasal influenza vaccine also produced significant cross immunity to five other H3N2 influenza virus strains not present in the vaccine and produced sterile immunity after challenge with homologous live virus. No safety issues were observed in 249 ferrets receiving the adjuvanted influenza vaccine. These findings demonstrate the ability of W₈₀5EC NE to adjuvant nasally administered influenza vaccine and provide a basis for studying the intranasal W₈₀5EC-adjuvanted influenza vaccine in humans.     

Immunity following intranasal administration of an inactivated,freeze-dried A/England/42/72 vaccine

A group of 23 student volunteers were each inoculated intranasally with 400 IU of inactivated, freeze-dried A/England/42/72 vaccine. Only one volunteer showed a four-fold rise in serum HI antibody following immunization, and the mean increase in serum HI antibody (gmt) for all volunteers did not increase two-fold. Thirteen of the volunteers developed detectable levels of nasal wash neutralizing antibody after immunization; local antibody was most commonly found in volunteers who produced a detectable but less than four-fold fise in serum antibody titre, and who produced nasal washings with relatively high concentrations of protein and secretory IgA. Four weeks after immunization, the vaccinees and a matched group of control subjects were inoculated with attenuated A/England/42/72 (MRC-7) virus. Evidence of infection was found in 14 of 23 (61 per cent) of control subjects and in seven of 23 (30 per cent) of immunized volunteers. This result showed a significant protection (P = 0.04) against challenge virus infection for volunteers given intranasal vaccine.     

Vaccination against H9N2 avian influenza virus reduces bronchus‐associated lymphoid tissue formation in cynomolgus macaques after intranasal virus challenge infection

H9N2 avian influenza virus causes sporadic human infection. Since humans do not possess acquired immunity specific to this virus, we examined the pathogenicity of an H9N2 virus isolated from a human and then analyzed protective effects of a vaccine in cynomolgus macaques. After intranasal challenge with A/Hong Kong/1073/1999 (H9N2) (HK1073) isolated from a human patient, viruses were isolated from nasal and tracheal swabs in unvaccinated macaques with mild fever and body weight loss. A formalin‐inactivated H9N2 whole particle vaccine derived from our virus library was subcutaneously inoculated to macaques. Vaccination induced viral antigen‐specific IgG and neutralization activity in sera. After intranasal challenge with H9N2, the virus was detected only the day after inoculation in the vaccinated macaques. Without vaccination, many bronchus‐associated lymphoid tissues (BALTs) were formed in the lungs after infection, whereas the numbers of BALTs were smaller and the cytokine responses were weaker in the vaccinated macaques than those in the unvaccinated macaques. These findings indicate that the H9N2 avian influenza virus HK1073 is pathogenic in primates but seems to cause milder symptoms than does H7N9 influenza virus as found in our previous studies and that a formalin‐inactivated H9N2 whole particle vaccine induces protective immunity against H9N2 virus.     

Comparison of intranasal and aerosol infection of mice in assessment of immunity to influenza virus infection.

A comparison was made of intranasal and aerosol routes of infection with X-31 influenza A virus in Balb/c mice. Mice were first infected with 100 MID50 by either route then challenged 42 days later with the same virus given by the same or alternative route. Three days following each infection, pulmonary virus was measured by inoculation of chick embryos. Mice initially infected under ether anesthesia by intranasal inoculation experienced higher initial mortality but proved most resistant to subsequent challenge by either method. In contrast, mice first infected by aerosol were least resistant to intranasal challenge, as indicated by increased rate of infection and pulmonary virus titers, but, like mice previously infected intranasally, were not reinfected by the aerosol route. Thus, intranasal infection appears to be more effective both in inducing and challenging immunity from infection. These results should be considered in the design of experiments utilizing influenza virus infection of mice as a model system.     

Rubella Immunization of Volunteers Via the Respiratory Tract

The efficacy of various routes of administration of the live attenuated rubella virus vaccine was evaluated by using 46 seronegative volunteers who were divided into 4 vaccine groups: subcutaneous, nosedrops, spray into posterior oropharynx and nose using large particle aerosol, and inhalation of small particle aerosol through the mouth. Seroconversion was observed in all of the vaccinees regardless of route of immunization. Nasal secretion antibody 6 weeks after immunization was highest in the volunteers who received the vaccine by nose drops (all members of this group had demonstrable nasal secretion antibody after immunization). Only half of the volunteers in the subcutaneous group developed demonstrable nasal secretion antibody. This suggests that nasal secretion antibody was best stimulated when vaccine was given directly into the nose. Volunteers were challenged with the vaccine intranasally at 6 to 8 weeks. None of the volunteers exhibited clinical symptoms or fourfold or greater serum antibody rises after challenge, but fourfold or greater nasal secretion antibody rises were observed in three volunteers in the subcutaneous vaccine group and two in the aerosol group, suggesting that those volunteers had not been protected against challenge. Rubella virus was isolated 8 to 12 days after challenge in two persons in the subcutaneous group and three in the aerosol vaccine group, but none in the nose drops or spray groups. Thus, protection after nasal challenge appeared to be best in those groups which also had the best nasal secretion antibody response after immunization. However, protection did not seem to be correlated with either nasal secretion or serum antibody levels.